Alpha-amylase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to variants of a parent alpha-amylase. The present invention also relates to polynucleotides encoding the variants and to nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of using the variant enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/137,852filed Apr. 25, 2016, now allowed, which is a divisional of U.S.application Ser. No. 13/515,437 filed Jun. 20, 2012, now U.S. Pat. No.9,416,355, which is a 35 U.S.C. 371 national application ofPCT/EP2011/050074 filed Jan. 4, 2011, which claims priority or thebenefit under 35 U.S.C. 119 of European application nos. 10150062.7 and10150063.5 both filed Jan. 4, 2010 and U.S. provisional application Nos.61/292,324, 61/292,327 61/304,092, 61/333,930, 61/354,775, 61/354,817,61/355,230 and 61/362,536 filed Jan. 5, 2010, Jan. 5, 2010, Feb. 12,2010, May 12, 2010, Jun. 15, 2010, Jun. 15, 2010, Jun. 16, 2010 and Jul.8, 2010, respectively, the contents of which are fully incorporatedherein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to alpha-amylase variants having animproved property, e.g., improved stability, polynucleotides encodingthe variants, methods of producing the variants, and methods of usingthe variants.

Description of the Related Art

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1)constitute a group of enzymes, which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides.

Alpha-amylases are used commercially for a variety of purposes such asin the initial stages of starch processing (e.g., liquefaction); in wetmilling processes; and in alcohol production from carbohydrate sources.They are also used as cleaning agents or adjuncts in detergent matrices;in the textile industry for starch desizing; in baking applications; inthe beverage industry; in oil fields in drilling processes; in recyclingprocesses, e.g., for de-inking paper; and in animal feed.

SUMMARY OF THE INVENTION

The present invention provides alpha-amylase variants with improvedproperties, e.g., improved thermostability, compared to their parentenzyme.

The present invention relates to isolated variants of a parentalpha-amylase, comprising a substitution at three or more (several)positions corresponding to positions 59, 89, 91, 96, 108, 112, 129, 157,165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254,269, 270, 274, 276, 281, 284, 416, and 427.

The present invention also relates to isolated polynucleotides encodingan alpha-amylase variant, nucleic acid constructs, vectors, and hostcells comprising the polynucleotides, and methods of producing a variantof a parent alpha-amylase.

The present invention also relates to the use of the variants in starchprocessing (e.g., liquefaction); wet milling processes; alcoholproduction from carbohydrate sources; detergents; dishwashingcompositions; starch desizing in the textile industry; bakingapplications; the beverage industry; oil fields in drilling processes;recycling processes, e.g., for de-inking paper, and animal feed.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1(a)-1(b) show an alignment of the catalytic domains ofalpha-amylases with the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5and 7. SEQ ID NO: 1 is a Bacillus stearothermophilus alpha-amylase; SEQID NO: 2 is Bacillus flavothermus amylase, AMY1048 described in WO2005/001064; SEQ ID NO: 3 is the Bacillus alpha-amylase TS-22; SEQ IDNO:4 is the Bacillus alpha-amylase TS-23 described in J. Appl.Microbiology, 1997, 82: 325-334 (SWALL:q59222); SEQ ID NO: 5 is analpha-amylase described in WO 2004/091544; and SEQ ID NO: 7 is anotherBacillus stearothermophilus alpha-amylase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated variants of a parentalpha-amylase, comprising a substitution at three or more (several)positions corresponding to positions 59, 89, 91, 96, 108, 112, 129, 157,165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254,269, 270, 274, 276, 281, 284, 416, and 427, wherein the variant has atleast 65% and less than 100% sequence identity with at least one of themature polypeptide of SEQ ID NO: 1; and the variant has alpha-amylaseactivity.

Definitions

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) area group of enzymes, which catalyze the hydrolysis of starch and otherlinear and branched 1,4-glucosidic oligo- and polysaccharides.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of its polypeptideproduct. The boundaries of the coding sequence are generally determinedby an open reading frame, which usually begins with the ATG start codonor alternative start codons such as GTG and TTG and ends with a stopcodon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a variant ofthe present invention. Each control sequence may be native or foreign tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parentalpha-amylase. Such improved properties include, but are not limited to,altered temperature-dependent activity profile, thermostability, pHactivity, pH stability, substrate specificity, product specificity, andchemical stability.

Isolated variant: The terms “isolated” and “purified” mean a polypeptideor polynucleotide that is removed from at least one component with whichit is naturally associated. For example, a variant may be at least 1%pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, atleast 40% pure, at least 60% pure, at least 80% pure, and at least 90%pure, as determined by SDS-PAGE and a polynucleotide may be at least 1%pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, atleast 40% pure, at least 60% pure, at least 80% pure, and at least 90%pure, as determined by agarose electrophoresis.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 483, 1 to 486, or 1 to 493 of SEQ IDNO: 1. It is known in the art that a host cell may produce a mixture oftwo or more different mature polypeptides (i.e., with a differentC-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a nucleotide sequence that encodes a mature polypeptidehaving alpha-amylase activity.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of the polynucleotide sequence such that thecontrol sequence directs the expression of the coding sequence of apolypeptide.

Parent: The term “parent” alpha-amylase means an alpha-amylase to whichan alteration is made to produce a variant of the present invention. Theparent may be a naturally occurring (wild-type) polypeptide or a variantthereof, prepared by suitable means. The parent may also be an allelicvariant.

Polypeptide fragment: The term “polypeptide fragment” means apolypeptide having one or more (several) amino acids deleted from theamino and/or carboxyl terminus of a mature polypeptide; wherein thefragment has alpha-amylase activity. In one aspect, a fragment containsat least 483 amino acid residues, e.g., at least 486 and at least 493amino acid residues, of the mature polypeptide of SEQ ID NO: 1.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide sequencehaving one or more (several) nucleotides deleted from the 5′ and/or 3′end of a mature polypeptide coding sequence; wherein the subsequenceencodes a polypeptide fragment having alpha-amylase activity.

Variant: The term “variant” means a polypeptide having alpha-amylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, of one or more (several) amino acid residues at one ormore (several) positions. A substitution means a replacement of an aminoacid occupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding 1-5 amino acids adjacent to and following an amino acid occupyinga position.

Wild-Type: The term “wild-type” means an alpha-amylase expressed by anaturally occurring microorganism, such as a bacterium, yeast, orfilamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 1 is used to determine the corresponding amino acidresidue in another alpha-amylase. The amino acid sequence of anotheralpha-amylase is aligned with the mature polypeptide disclosed in SEQ IDNO: 1, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 1 can be determined determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater.

Identification of the corresponding amino acid residue in anotheralpha-amylase can be confirmed by an alignment of multiple polypeptidesequences using “ClustalW” (Larkin et al., 2007, Bioinformatics 23:2947-2948).

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 1 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more (several) representatives in the protein structure databases.Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815;McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilizeinformation from a variety of sources (PSI-BLAST, secondary structureprediction, structural alignment profiles, and solvation potentials) asinput to a neural network that predicts the structural fold for a querysequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol.313: 903-919, can be used to align a sequence of unknown structure withthe superfamily models present in the SCOP database. These alignmentscan in turn be used to generate homology models for the polypeptide, andsuch models can be assessed for accuracy using a variety of toolsdeveloped for that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Eng. 11: 739-747), and implementations of these algorithms canadditionally be utilized to query structure databases with a structureof interest in order to discover possible structural homologs (e.g.,Holm and Park, 2000, Bioinformatics 16: 566-567). These structuralalignments can be used to predict the structurally and functionallycorresponding amino acid residues in proteins within the same structuralsuperfamily. This information, along with information derived fromhomology modeling and profile searches, can be used to predict whichresidues to mutate when moving mutations of interest from one protein toa close or remote homolog.

In describing the alpha-amylase variants of the present invention, thenomenclature described below is adapted for ease of reference. In allcases, the accepted IUPAC single letter or triple letter amino acidabbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine with alanine at position 226 is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representingmutations at positions 205 and 411 substituting glycine (G) witharginine (R), and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:original amino acid, position, original amino acid, new inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. Multiple insertions of aminoacids are designated [Original amino acid, position, original aminoacid, new inserted amino acid #1, new inserted amino acid #2; etc.]. Forexample, the insertion of lysine and alanine after glycine at position195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of tyrosine and glutamic acid for arginine and glycine atpositions 170 and 195, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginge with tyrosine or glutamic acid atposition 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants:

Tyr167Gly+Arg170Gly, Tyr167Gly+Arg170Ala, Tyr167Ala+Arg170Gly, andTyr167Ala+Arg170Ala. Parent Alpha-Amylases

In a first aspect, the parent alpha-amylase has a sequence identity tothe mature polypeptide of SEQ ID NO: 1 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

In a second aspect, the parent alpha-amylase has a sequence identity tothe mature polypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

In a third aspect, the parent alpha-amylase has a sequence identity tothe mature polypeptide of SEQ ID NO: 3 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

In a fourth aspect, the parent alpha-amylase has a sequence identity tothe mature polypeptide of SEQ ID NO: 4 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

In a fifth aspect, the parent alpha-amylase has a sequence identity tothe mature polypeptide of SEQ ID NO: 5 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

In a sixth aspect, the parent alpha-amylase has a sequence identity tothe mature polypeptide of SEQ ID NO: 6 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

In a seventh aspect, the parent alpha-amylase has a sequence identity tothe mature polypeptide of SEQ ID NO: 7 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

The parent alpha-amylase preferably comprises the amino acid sequence ofSEQ ID NO: 1. In another aspect, the parent alpha-amylase comprises orconsists of the mature polypeptide of SEQ ID NO: 1. In another aspect,the parent alpha-amylase comprises or consists of amino acids 1 to 486or amino acids 1 to 493 of SEQ ID NO: 1.

The parent alpha-amylase preferably comprises the amino acid sequence ofSEQ ID NO: 2. In another aspect, the parent alpha-amylase comprises orconsists of the mature polypeptide of SEQ ID NO: 2. In another aspect,the parent alpha-amylase comprises or consists of amino acids 1 to 482of SEQ ID NO: 2. Amino acids 483-584 of SEQ ID NO: 2 represent a CBM20binding domain.

The parent alpha-amylase preferably comprises the amino acid sequence ofSEQ ID NO: 3. In another aspect, the parent alpha-amylase comprises orconsists of the mature polypeptide of SEQ ID NO: 3. In another aspect,the parent alpha-amylase comprises or consists of amino acids 1 to 484of SEQ ID NO: 3. Amino acids 485-586 of SEQ ID NO: 3 represent a CBM20binding domain.

The parent alpha-amylase preferably comprises the amino acid sequence ofSEQ ID NO: 4. In another aspect, the parent alpha-amylase comprises orconsists of the mature polypeptide of SEQ ID NO: 4. In another aspect,the parent alpha-amylase comprises or consists of amino acids 1 to 484of SEQ ID NO: 4. Amino acids 485-583 of SEQ ID NO: 4 represent a CBM20binding domain.

The parent alpha-amylase preferably comprises the amino acid sequence ofSEQ ID NO: 5. In another aspect, the parent alpha-amylase comprises orconsists of the mature polypeptide of SEQ ID NO: 5. In another aspect,the parent alpha-amylase comprises or consists of amino acids 1 to 484of SEQ ID NO: 5. Amino acids 485-583 of SEQ ID NO: 5 represent a CBM20binding domain.

The parent alpha-amylase preferably comprises or consists of the aminoacid sequence of SEQ ID NO: 6.

The parent alpha-amylase preferably comprises the amino acid sequence ofSEQ ID NO: 7. In another aspect, the parent alpha-amylase comprises orconsists of the mature polypeptide of SEQ ID NO: 7. In another aspect,the parent alpha-amylase comprises or consists of amino acids 1 to 486of SEQ ID NO: 7.

In an embodiment, the parent alpha-amylase is a fragment of the maturepolypeptide of SEQ ID NO: 1 containing at least 483 amino acid residues.

In an embodiment, the parent alpha-amylase is a fragment of the maturepolypeptide of SEQ ID NO: 2 containing at least 482 amino acid residues.

In an embodiment, the parent alpha-amylase is a fragment of the maturepolypeptide of SEQ ID NO: 3 containing at least 484 amino acid residues.

In an embodiment, the parent alpha-amylase is a fragment of the maturepolypeptide of SEQ ID NO: 4 containing at least 484 amino acid residues.

In an embodiment, the parent alpha-amylase is a fragment of the maturepolypeptide of SEQ ID NO: 5 containing at least 484 amino acid residues.

In an embodiment, the parent alpha-amylase is a fragment of the maturepolypeptide of SEQ ID NO: 6 containing at least 486 amino acid residues.

In an embodiment, the parent alpha-amylase is a fragment of the maturepolypeptide of SEQ ID NO: 7 containing at least 486 amino acid residues.

In another embodiment, the parent enzyme is an allelic variant of themature polypeptide of SEQ ID NO: 1.

In another embodiment, the parent enzyme is an allelic variant of themature polypeptide of SEQ ID NO: 2.

In another embodiment, the parent enzyme is an allelic variant of themature polypeptide of SEQ ID NO: 3.

In another embodiment, the parent enzyme is an allelic variant of themature polypeptide of SEQ ID NO: 4.

In another embodiment, the parent enzyme is an allelic variant of themature polypeptide of SEQ ID NO: 5.

In another embodiment, the parent enzyme is an allelic variant of themature polypeptide of SEQ ID NO: 6.

In another embodiment, the parent enzyme is an allelic variant of themature polypeptide of SEQ ID NO: 7.

The parent alpha-amylase may be obtained from microorganisms of anygenus. For purposes of the present invention, the term “obtained from”as used herein in connection with a given source shall mean that theparent alpha-amylase encoded by a polynucleotide is produced by thesource or by a cell in which the polynucleotide from the source has beeninserted. In one aspect, the parent alpha-amylase is secretedextracellularly.

The parent alpha-amylase may be a bacterial alpha-amylase. For example,the alpha-amylase may be a gram-positive bacterial polypeptide such as aBacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, orStreptomyces alpha-amylase, or a gram-negative bacterial polypeptidesuch as a Campylobacter, E. coli, Flavobacterium, Fusobacterium,Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, orUreaplasman alpha-amylase.

In one aspect, the parent alpha-amylase is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis alpha-aylase.

In another aspect, the parent alpha-amylase is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus alpha-amylase.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans alpha-amylase.

The parent alpha-amylase may be a fungal alpha-amylase. In anotheraspect, the fungal alpha-amylase is a yeast alpha-amylase such as aCandida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia alpha-amylase. In another aspect, the fungal alpha-amylase is afilamentous fungal alpha-amylase such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria alpha-amylase.

In another aspect, the parent alpha-amylase is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis alpha-amylase.

In another aspect, the parent alpha-amylase is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfoetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viridealpha-amylase.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent alpha-amylase may also be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) or DNA samples obtained directly from naturalmaterials (e.g., soil, composts, water, etc) using the above-mentionedprobes. Techniques for isolating microorganisms and DNA directly fromnatural habitats are well known in the art. The polynucleotide encodingan alpha-amylase may then be derived by similarly screening a genomic orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding an alpha-amylase has been detected with suitableprobe(s) as described herein, the sequence may be isolated or cloned byutilizing techniques that are known to those of ordinary skill in theart (see, e.g., Sambrook et al., 1989, supra).

The parent alpha-amylase can also include hybrid polypeptides in which aportion of one polypeptide is fused at the N-terminus or the C-terminusof a portion of another polypeptide.

The parent alpha-amylase can also include fused polypeptides orcleavable fusion polypeptides in which one polypeptide is fused at theN-terminus or the C-terminus of another polypeptide. A fused polypeptideis produced by fusing a polynucleotide (or a portion thereof) encodinganother polypeptide to a polynucleotide (or a portion thereof) of thepresent invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.Fusion proteins may also be constructed using intein technology in whichfusions are created post-translationally (Cooper et al., 1993, EMBO J.12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thevariant from the fusion protein. Examples of cleavage sites include, butare not limited to, the cleavage sites disclosed in Martin et al., 2003,J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J.Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ.Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503;Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986,Biochem. 25: 505-512); Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, andGenetics 6: 240-248); and Stevens, 2003, Drug Discovery World 4: 35-48.

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving alpha-amylase activity, comprising: (a) introducing into a parentalpha-amylase a substitution at three or more (several) positionscorresponding to positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166,168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270,274, 276, 281, 284, 416, and 427, wherein the variant has alpha-amylaseactivity; and (b) recovering the variant.

The variants can be prepared according to any mutagenesis procedureknown in the art, such as site-directed mtagenesis, synthetic geneconstruction, semi-synthetic gene construction, random mutagenesis,shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (several)mutations are created at a defined site in a polynucleotide moleculeencoding the parent alpha-amylase. The technique can be performed invitro or in vivo.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide molecule of interest.Gene synthesis can be performed utilizing a number of techniques, suchas the multiplex microchip-based technology described by Tian et al.,2004, Nature 432: 1050-1054, and similar technologies whereinolgionucleotides are synthesized and assembled upon photo-programmablemicrofluidic chips.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parentalpha-amylase and subsequent ligation of an oligonucleotide containingthe mutation in the polynucleotide. Usually the restriction enzyme thatdigests at the plasmid and the oligonucleotide is the same, permittingsticky ends of the plasmid and insert to ligate to one another. See, forexample, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Research 18:7349-4966.

Site-directed mutagenesis can be accomplished in vivo by methods knownin the art. See, for example, U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnology 19: 773-776;Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants of a parent alpha-amylase.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide fragments may then be shuffled.

Variants

The present invention relates to variants comprising a substitution atthree or more (several) positions corresponding to positions 59, 89, 91,96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184,208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and 4427,wherein the variant having alpha-amylase activity.

In an embodiment, the variant has a sequence identity of at least 65%,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, to the amino acid sequence of the parentalpha-amylase.

In another embodiment, the variant has a sequence identity of at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, andat least 99%, but less than 100% with the mature polypeptide of SEQ IDNO: 1.

In another embodiment, the variant has a sequence identity of at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, andat least 99%, but less than 100% with the mature polypeptide of SEQ IDNO: 2.

In another embodiment, the variant has a sequence identity of at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, andat least 99%, but less than 100% with the mature polypeptide of SEQ IDNO: 3.

In another embodiment, the variant has a sequence identity of at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, andat least 99%, but less than 100% with the mature polypeptide of SEQ IDNO: 4.

In another embodiment, the variant has a sequence identity of at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, andat least 99%, but less than 100% with the mature polypeptide of SEQ IDNO: 5.

In another embodiment, the variant has a sequence identity of at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, andat least 99%, but less than 100% with the mature polypeptide of SEQ IDNO: 6.

In another embodiment, the variant has a sequence identity of at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, andat least 99%, but less than 100% with the mature polypeptide of SEQ IDNO: 7.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 59 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, inparticular with Ala, Gln, Glu, Gly, Ile, Leu, Prot, or Thr.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 89 with Ala, Arg, Asn, Asp, Cys, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Arg, His, or Lys.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 91 with Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ile, Leu, or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 96 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ile, Leu, or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 108 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ala.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 112 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ala, Asp, or Glu.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 129 with Ala, Arg, Asn, Asp, Cys, Gin, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ala, Thr, or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 157 with Ala, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with His, Lys, Phe, or Tyr.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 165 with Ala, Arg, Asn, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Asn.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 166 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val, inparticular with Phe.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 168 with Ala, Arg, Asn, Asp, Cys, Gin, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ala.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 171 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Glu.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 177 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Arg, Leu, or Met.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 179 with Ala, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Gin, Glu, Ile, Leu, Lys, or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 180 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Glu or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 181 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Glu, Gly, or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 184 with Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ser.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 208 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Phe or Tyr.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 220 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Pro.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 224 with Ala, Arg, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Leu.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 242 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, inparticular with Ala, Asp, Glu, Gin, or Met.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 254 with Ala, Arg, Asn, Asp, Cys, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ala, Ser, or Thr.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 269 with Ala, Arg, Asn, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Gin or Glu.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 270 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Leu, Thr, or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 274 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Arg, Gin, Glu, Lys, or Phe.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 276 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, inparticular with Phe.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 281 with Ala, Arg, Asn, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Asn or Ser.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 284 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with His, Thr, or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 416 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ala, Asn, Asp, Ser, Thr, or Val.

In an embodiment, the variant comprises a substitution at a positioncorresponding to position 427 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ile, Met, or Val.

In an aspect, the variant comprising a substitution at three positionscorresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157,165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254,269, 270, 274, 276, 281, 284, 416, and 427. In particular, the variantcomprises a substitution at the positions corresponding to positions129, 177, and 179 or positions 220, 242, and 254.

In another aspect, the variant comprises a substitution at fourpositions corresponding to any of positions 59, 89, 91, 96, 108, 112,129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224,242, 254, 269, 270, 274, 276, 281, 284, 416, and 427. In particular, thevariant comprises a substitution at the positions corresponding topositions 129, 177, 179, and 208; positions 129, 177, 179, and 242;positions 129, 177, 179, and 284; positions 208, 220, 224, and 254;positions 220, 224, 242, and 254; or positions 220, 224, 254, and 284.

In another aspect, the variant comprises a substitution at fivepositions corresponding to any of positions 59, 89, 91, 96, 108, 112,129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224,242, 254, 269, 270, 274, 276, 281, 284, 416, and 427. In particular, thevariant comprises a substitution at the positions corresponding topositions 129, 177, 179, 208, and 242; positions 129, 177, 179, 208, and284; positions 208, 220, 224, 242, and 254; or positions 208, 220, 224,254, and 284.

In another aspect, the variant comprises a substitution at six positionscorresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157,165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254,269, 270, 274, 276, 281, 284, 416, and 427. In particular, the variantcomprises a substitution at the positions corresponding to positions129, 177, 179, 208, 242, and 284 or positions 129, 177, 179, 220, 224,and 254.

In another aspect, the variant comprises a substitution at sevenpositions corresponding to any of positions 59, 89, 91, 96, 108, 112,129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224,242, 254, 269, 270, 274, 276, 281, 284, 416, and 427.

In another aspect, the variant comprises a substitution at eightpositions corresponding to any of positions 59, 89, 91, 96, 108, 112,129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224,242, 254, 269, 270, 274, 276, 281, 284, 416, and 427.

In another aspect, the variant comprises a substitution at ninepositions corresponding to any of positions 59, 89, 91, 96, 108, 112,129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224,242, 254, 269, 270, 274, 276, 281, 284, 416, and 427.

In another aspect, the variant comprises a substitution at ten positionscorresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157,165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254,269, 270, 274, 276, 281, 284, 416, and 427.

The variants preferably have 3-20, e.g., 3-10 and 6-10, alterations suchas 3, 4, 5, 6, 7, 8, 9 or 10 alterations. In an embodiment, thealterations are substitutions.

In an embodiment, the variant alpha-amylases comprise or consist of aset of substitutions selected from the group consisting of:

V59A+G108A;

S242Q+M284V;

V59A+M284V;

G108A+M284V;

V59A+G108A+M284V;

V59A+G108A+S242Q+M284V;

E129V+K177L+R179E;

K220P+N224L+Q254S;

E129V+K177L+R179E+M284V;

V59A+E129V+K177L+R179E+H208Y+M284V;

V59A+H208Y+K220P+N224L+Q254S+M284V;

E129V+K177L+R179E+K220P+N224L+Q254S;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S+M284V;

V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;

V59A+Q89R+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S+M284V; and

V59A+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V.

The variants may further comprise a deletion at one or more, e.g., two,three or four, positions corresponding to positions 179, 180, 181 and182. For example, the variants may comprise a deletion at positionscorresponding to positions 181 and 182.

The variants also may further comprise an alteration, preferably asubstitution, at a position corresponding to position 193. For example,the alteration at a position corresponding to position 193 may be asubstitution with Phe.

The variants also may further comprise a deletion of the amino acid atthe position corresponding to positions 376 and/or 377.

In another aspect, the variants further comprise an additionalalteration, preferably a substitution, at a position corresponding toposition 200, such as a substitution at a position corresponding toposition 200 with Leu, Ile, or Thr.

The variants of the present invention preferably consist of 483 to 515,483 to 493, or 483 to 486 amino acids.

Polynucleotides

The present invention also relates to isolated polynucleotides thatencode any of the variants of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more (several) control sequences that direct theexpression of the coding sequence in a suitable host cell underconditions compatible with the control sequences.

An isolated polynucleotide encoding a variant may be manipulated in avariety of ways to provide for expression of the variant. Manipulationof the polynucleotide prior to its insertion into a vector may bedesirable or necessary depending on the expression vector. Thetechniques for modifying polynucleotides utilizing recombinant DNAmethods are well known in the art.

The control sequence may be a promoter sequence, which is recognized bya host cell for expression of the polynucleotide. The promoter sequencecontains transcriptional control sequences that mediate the expressionof the variant. The promoter may be any nucleic acid sequence that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformisalpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene(penP), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xyIA andxyIB genes, E. coli lac operon, Streptomyces coelicolor agarase gene(dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al.,1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25).Further promoters are described in “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242: 74-94; and in Sambrook etal., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter including a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the variant. Any terminator that is functionalin the host cell may be used in the present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′-terminus ofthe polynucleotide encoding the variant. Any leader sequence that isfunctional in the host cell may be used in the present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polypeptide-encoding sequenceand, when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of avariant and directs the encoded polypeptide into the cell's secretorypathway. The 5′-end of the coding sequence of the polynucleotide mayinherently contain a signal peptide coding region naturally linked intranslation reading frame with the segment of the coding region thatencodes the secreted variant. Alternatively, the 5′-end of the codingsequence may contain a signal peptide coding region that is foreign tothe coding sequence. The foreign signal peptide coding region may berequired where the coding sequence does not naturally contain a signalpeptide coding region. Alternatively, the foreign signal peptide codingregion may simply replace the natural signal peptide coding region inorder to enhance secretion of the variant. However, any signal peptidecoding region that directs the expressed polypeptide into the secretorypathway of a host cell may be used in the present invention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of avariant. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Myceliophthora thermophila laccase (WO 95/33836), Rhizomucormiehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the variant relative to the growth ofthe host cell. Examples of regulatory systems are those that cause theexpression of the gene to be turned on or off in response to a chemicalor physical stimulus, including the presence of a regulatory compound.Regulatory systems in prokaryotic systems include the lac, tac, and trpoperator systems. In yeast, the ADH2 system or GAL1 system may be used.In filamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding the variantwould be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences described above may be joinedtogether to produce a recombinant expression vector that may include oneor more (several) convenient restriction sites to allow for insertion orsubstitution of the polynucleotide encoding the variant at such sites.Alternatively, the polynucleotide may be expressed by inserting thepolynucleotide or a nucleic acid construct comprising the polynucleotideinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the polynucleotide. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors of the present invention preferably contain one or more(several) selectable markers that permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAM111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of an alpha-amylasevariant. An increase in the copy number of the polynucleotide can beobtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the polynucleotide where cells containing amplifiedcopies of the selectable marker gene, and thereby additional copies ofthe polynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra) to obtain substantially pure alpha-amylase variants.

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant, which are advantageously used inthe recombinant production of the variant. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. Thechoice of a host cell will to a large extent depend upon the geneencoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive bacterium orgram-negative bacterium. Gram-positive bacteria include, but are notlimited to, Bacillus, Clostridium, Enterococcus, Geobacillus,Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,Streptococcus, and Streptomyces. Gram-negative bacteria include, but arenot limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium,Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, andUreaplasma.

The bacterial host cell may be any Bacillus cell, including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell, including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-2070, by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In one aspect, the host cell is a fungal cell. “Fungi” as used hereinincludes the phyla Ascomycota, Basidiomycota, Chytridiomycota, andZygomycota as well as the Oomycota and all mitosporic fungi (as definedby Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi,8th edition, 1995, CAB International, University Press, Cambridge, UK).

In another aspect, the fungal host cell is a yeast cell. “Yeast” as usedherein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In another aspect, the yeast host cell is a Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiacell.

In another aspect, the yeast host cell is a Kluyveromyces lactis,Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolyticacell.

In another aspect, the fungal host cell is a filamentous fungal cell.“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).The filamentous fungi are generally characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

In another aspect, the filamentous fungal host cell is an Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In another aspect, the filamentous fungal host cell is an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for the expression of the variant; and (b)recovering the variant from the cultivation medium.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing a variant is used as asource of the variant.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods may include use ofspecific antibodies, formation of an enzyme product, or disappearance ofan enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein in theExamples.

The variant may be recovered by methods known in the art. For example,the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

A variant of the present invention may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure variants.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a variant of the present invention so as to express and producethe variant in recoverable quantities. The variant may be recovered fromthe plant or plant part. Alternatively, the plant or plant partcontaining the recombinant variant may be used as such for improving thequality of a food or feed, e.g., improving nutritional value,palatability, and rheological properties, or to destroy an antinutritivefactor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant of the presentinvention may be constructed in accordance with methods known in theart. In short, the plant or plant cell is constructed by incorporatingone or more (several) expression constructs encoding a polypeptide ofthe present invention into the plant host genome or chloroplast genomeand propagating the resulting modified plant or plant cell into atransgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant of the present inventionoperably linked with appropriate regulatory sequences required forexpression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant is desired tobe expressed. For instance, the expression of the gene encoding avariant may be constitutive or inducible, or may be developmental, stageor tissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiol. 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000, the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), or thealdP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can alsobe used for transforming monocots, although other transformation methodsare often used for these plants. Presently, the method of choice forgenerating transgenic monocots is particle bombardment (microscopic goldor tungsten particles coated with the transforming DNA) of embryoniccalli or developing embryos (Christou, 1992, Plant J. 2: 275-281;Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674). An alternative method for transformation ofmonocots is based on protoplast transformation as described by Omirullehet al., 1993, Plant Molecular Biology 21: 415-428. Additionaltransformation methods for use in accordance with the present disclosureinclude those described in U.S. Pat. Nos. 6,395,966 and 7,151,204 (bothof which are herein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having a construct of the presentinvention to a second plant lacking the construct. For example, aconstruct encoding a variant or a portion thereof can be introduced intoa particular plant variety by crossing, without the need for everdirectly transforming a plant of that given variety. Therefore, thepresent invention not only encompasses a plant directly regenerated fromcells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention, or aportion of a DNA construct prepared in accordance with the presentinvention. Crossing results in a transgene of the present inventionbeing introduced into a plant line by cross pollinating a starting linewith a donor plant line that includes a transgene of the presentinvention. Non-limiting examples of such steps are further articulatedin U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a variant ofthe present invention comprising: (a) cultivating a transgenic plant ora plant cell comprising a polynucleotide encoding a variant of thepresent invention under conditions conducive for production of thevariant; and (b) recovering the variant.

Compositions

The present invention also relates to compositions comprising analpha-amylase variant and at least one additional enzyme. The additionalenzyme(s) may be selected from the group consisting of beta-amylase,cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase),glucoamylase, hemicellulsae (e.g., xylanase), isoamylase, isomerase,lipase, phytase, protease, pullulanase, and/or other enzymes useful in acommercial process in conjunction with an alpha-amylase. The additionalenzyme may also be a second alpha-amylase. Such enzymes are known in theart in starch processing, sugar conversion, fermentations for alcoholand other useful end-products, commercial detergents and cleaning aids,stain removal, fabric treatment or desizing, and the like.

Methods of Using the Alpha-Amylase Variants—Industrial Applications

The variants of the present invention possess valuable propertiesallowing for a variety of industrial applications. In particular, thevariants may be used in detergents, in particular laundry detergentcompositions and dishwashing detergent compositions, hard surfacecleaning compositions, and for desizing textiles, fabrics or garments,production of pulp and paper, beer making, ethanol production, andstarch conversion processes.

The alpha-amylase variants may be used for starch processes, inparticular starch conversion, especially liquefaction of starch (see,e.g., U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, WO 99/19467, andWO 96/28567, which are all hereby incorporated by reference). Alsocontemplated are compositions for starch conversion purposes, which maybeside the variant of the invention also comprise an AMG, pullulanase,and other alpha-amylases.

Further, the variants are particularly useful in the production ofsweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017, which ishereby incorporated by reference), such as fuel, drinking and industrialethanol, from starch or whole grains.

The variants may also be used for desizing of textiles, fabrics, andgarments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP119920, which are incorporated herein by reference), beer making orbrewing, and in pulp and paper production or related processes.

Starch Processing

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be suitably processed. This reduction in viscosity isprimarily attained by enzymatic degradation in current commercialpractice.

Conventional starch-conversion processes, such as liquefaction andsaccharification processes are described, e.g., in U.S. Pat. No.3,912,590, EP 252730 and EP 063909, which are incorporated herein byreference.

In an embodiment, the conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step.

In the case of converting starch into a sugar, the starch isdepolymerized. Such a depolymerization process consists of, e.g., apre-treatment step and two or three consecutive process steps, i.e., aliquefaction process, a saccharification process, and depending on thedesired end-product, an optional isomerization process.

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process, the pH is increased to a value in the range of6-8, e.g., pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immobilized glucose isomerase.

Production of Fermentation Products

In general, alcohol production (ethanol) from whole grain can beseparated into 4 main steps: milling, liquefaction, saccharification,and fermentation.

The grain is milled in order to open up the structure and allow forfurther processing. Two processes used are wet or dry milling. In drymilling, the whole kernel is milled and used in the remaining part ofthe process. Wet milling gives a very good separation of germ and meal(starch granules and protein) and is with a few exceptions applied atlocations where there is a parallel production of syrups.

In the liquefaction process the starch granules are solubilized byhydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysismay be carried out by acid treatment or enzymatically by analpha-amylase. Acid hydrolysis is used on a limited basis. The rawmaterial can be milled whole grain or a side stream from starchprocessing.

During a typical enzymatic liquefaction, the long-chained starch isdegraded into branched and linear shorter units (maltodextrins) by analpha-amylase. Enzymatic liquefaction is generally carried out as athree-step hot slurry process. The slurry is heated to between 60-95° C.(e.g., 77-86° C., 80-85° C., or 83-85° C.) and the enzyme(s) is (are)added. The liquefaction process is carried out at 85° C. for 1-2 hours.The pH is generally between 5.5 and 6.2. In order to ensure optimalenzyme stability under these conditions, 1 mM of calcium is added (toprovide about 40 ppm free calcium ions). After such treatment, theliquefied starch will have a “dextrose equivalent” (DE) of 10-15.

The slurry is subsequently jet-cooked at between 95-140° C., e.g.,105-125° C., cooled to 60-95° C. and more enzyme(s) is (are) added toobtain the final hydrolysis. The liquefaction process is carried out atpH 4.5-6.5, typically at a pH between 5 and 6. Milled and liquefiedgrain is also known as mash.

Liquefied starch-containing material is saccharified in the presence ofsaccharifying enzymes such as glucoamylases. The saccharificationprocess may last for 12 hours to 120 hours (e.g., 12 to 90 hours, 12 to60 hours and 12 to 48 hours).

However, it is common to perform a pre-saccharification step for about30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to65° C., typically around 60° C. which is followed by a completesaccharification during fermentation referred to as simultaneoussaccharification and fermentation (SSF). The pH is usually between4.2-4.8, e.g., pH 4.5. In a simultaneous saccharification andfermentation (SSF) process, there is no holding stage forsaccharification, rather, the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced duringliquefaction are converted into dextrose by adding a glucoamylase and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase. The temperature is lowered to 60° C., prior to the additionof the glucoamylase and debranching enzyme. The saccharification processproceeds for 24-72 hours.

Prior to addition of the saccharifying enzymes, the pH is reduced tobelow 4.5, while maintaining a high temperature (above 95° C.), toinactivate the liquefying alpha-amylase. This process reduces theformation of short oligosaccharide called “panose precursors,” whichcannot be hydrolyzed properly by the debranching enzyme. Normally, about0.2-0.5% of the saccharification product is the branched trisaccharidepanose (Glc pα1-6Glc pα1-4Glc), which cannot be degraded by apullulanase. If active amylase from the liquefaction remains presentduring saccharification (i.e., no denaturing), the amount of panose canbe as high as 1-2%, which is highly undesirable since it lowers thesaccharification yield significantly.

Fermentable sugars (e.g., dextrins, monosaccharides, particularlyglucose) are produced from enzymatic saccharification. These fermentablesugars may be further purified and/or converted to useful sugarproducts. In addition, the sugars may be used as a fermentationfeedstock in a microbial fermentation process for producingend-products, such as alcohol (e.g., ethanol and butanol), organic acids(e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol),ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).

In an embodiment, the fermentable sugars obtained during theliquefaction process steps are used to produce alcohol and particularlyethanol. In ethanol production, an SSF process is commonly used whereinthe saccharifying enzymes and fermenting organisms (e.g., yeast) areadded together and then carried out at a temperature of 30-40° C.

The organism used in fermentation will depend on the desiredend-product. Typically, if ethanol is the desired end product yeast willbe used as the fermenting organism. In some preferred embodiments, theethanol-producing microorganism is a yeast and specificallySaccharomyces such as strains of S. cerevisiae (U.S. Pat. No.4,316,956). A variety of S. cerevisiae are commercially available andthese include but are not limited to FALI (Fleischmann's Yeast),SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) andAngel alcohol yeast (Angel Yeast Company, China). The amount of starteryeast employed in the methods is an amount effective to produce acommercially significant amount of ethanol in a suitable amount of time,(e.g., to produce at least 10% ethanol from a substrate having between25-40% DS in less than 72 hours). Yeast cells are generally supplied inamounts of about 10⁴ to about 10¹², and preferably from about 10⁷ toabout 10¹⁰ viable yeast count per mL of fermentation broth. After yeastis added to the mash, it is typically subjected to fermentation forabout 24-96 hours, e.g., 35-60 hours. The temperature is between about26-34° C., typically at about 32° C., and the pH is from pH 3-6, e.g.,around pH 4-5.

The fermentation may include, in addition to a fermenting microorganisms(e.g., yeast), nutrients, and additional enzymes, including phytases.The use of yeast in fermentation is well known in the art.

In further embodiments, use of appropriate fermenting microorganisms, asis known in the art, can result in fermentation end product including,e.g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lacticacid, amino acids, and derivatives thereof. More specifically whenlactic acid is the desired end product, a Lactobacillus sp. (L. casei)may be used; when glycerol or 1,3-propanediol are the desiredend-products E. coli may be used; and when 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired endproducts, Pantoea citrea may be used as the fermenting microorganism.The above enumerated list are only examples and one skilled in the artwill be aware of a number of fermenting microorganisms that may be usedto obtain a desired end product.

Processes for Producing Fermentation Products from UngelatinizedStarch-Containing Material

The invention relates to processes for producing fermentation productsfrom starch-containing material without gelatinization (i.e., withoutcooking) of the starch-containing material. The fermentation product,such as ethanol, can be produced without liquefying the aqueous slurrycontaining the starch-containing material and water. In one embodiment aprocess of the invention includes saccharifying (e.g., milled)starch-containing material, e.g., granular starch, below the initialgelatinization temperature, preferably in the presence of alpha-amylaseand/or carbohydrate-source generating enzyme(s) to produce sugars thatcan be fermented into the fermentation product by a suitable fermentingorganism. In this embodiment the desired fermentation product, e.g.,ethanol, is produced from ungelatinized (i.e., uncooked), preferablymilled, cereal grains, such as corn. Accordingly, in the first aspectthe invention relates to processes for producing fermentation productsfrom starch-containing material comprising simultaneously saccharifyingand fermenting starch-containing material using a carbohydrate-sourcegenerating enzyme and a fermenting organism at a temperature below theinitial gelatinization temperature of said starch-containing material.In an embodiment a protease is also present. The protease may be anyacid fungal protease or metalloprotease. The fermentation product, e.g.,ethanol, may optionally be recovered after fermentation, e.g., bydistillation. Typically amylase(s), such as glucoamylase(s) and/or othercarbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are)present during fermentation. Examples of glucoamylases and othercarbohydrate-source generating enzymes include raw starch hydrolyzingglucoamylases. Examples of alpha-amylase(s) include acid alpha-amylasessuch as acid fungal alpha-amylases. Examples of fermenting organismsinclude yeast e.g., a strain of Saccharomyces cerevisiae. The term“initial gelatinization temperature” means the lowest temperature atwhich starch gelatinization commences. In general, starch heated inwater begins to gelatinize between about 50° C. and 75° C.; the exacttemperature of gelatinization depends on the specific starch and canreadily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material may be determined asthe temperature at which birefringence is lost in 5% of the starchgranules using the method described by Gorinstein and Lii, 1992,Starch/Starke 44(12): 461-466. Before initiating the process a slurry ofstarch-containing material, such as granular starch, having 10-55 w/w %dry solids (DS), preferably 25-45 w/w % dry solids, more preferably30-40 w/w % dry solids of starch-containing material may be prepared.The slurry may include water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants. Because the process of the invention iscarried out below the initial gelatinization temperature, and thus nosignificant viscosity increase takes place, high levels of stillage maybe used if desired. In an embodiment the aqueous slurry contains fromabout 1 to about 70 vol. %, preferably 15-60 vol. %, especially fromabout 30 to 50 vol. % water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants, or combinations thereof, or the like. Thestarch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids in the starch-containing material are convertedinto a soluble starch hydrolyzate. A process in this aspect of theinvention is conducted at a temperature below the initial gelatinizationtemperature, which means that the temperature typically lies in therange between 30-75° C., preferably between 45-60° C. In a preferredembodiment the process carried at a temperature from 25° C. to 40° C.,such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferablyaround 32° C. In an embodiment the process is carried out so that thesugar level, such as glucose level, is kept at a low level, such asbelow 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %,such as below about 1 w/w %, such as below about 0.5 w/w %, or below0.25 w/w %, such as below about 0.1 w/w %. Such low levels of sugar canbe accomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich doses/quantities of enzyme and fermenting organism to use. Theemployed quantities of enzyme and fermenting organism may also beselected to maintain low concentrations of maltose in the fermentationbroth. For instance, the maltose level may be kept below about 0.5 w/w%, such as below about 0.2 w/w %. The process of the invention may becarried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, ormore preferably from pH 4 to 5. In an embodiment fermentation is ongoingfor 6 to 120 hours, in particular 24 to 96 hours.

Processes for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect the invention relates to processes for producingfermentation products, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.Consequently, the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

(a) liquefying starch-containing material in the presence of analpha-amylase variant, or;

(b) saccharifying the liquefied material obtained in step (a) using acarbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

In an aspect, a pullulanase such as a family GH57 pullulanase is alsoused in the liquefaction step. In an embodiment a protease, such as anacid fungal protease or a metallo protease is added before, duringand/or after liquefaction. In an embodiment the metalloprotease isderived from a strain of Thermoascus, e.g., a strain of Thermoascusaurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670. In anembodiment the carbohydrate-source generating enzyme is a glucoamylasederived from a strain of Aspergillus, e.g., Aspergillus niger orAspergillus awamori, a strain of Talaromyces, especially Talaromycesemersonii; or a strain of Athelia, especially Athelia rolfsii; a strainof Trametes, e.g., Trametes cingulata; a strain of the genusPachykytospora, e.g., a strain of Pachykytospora papyracea; or a strainof the genus Leucopaxillus, e.g., Leucopaxillus giganteus; or a strainof the genus Peniophora, e.g., a strain of the species Peniophorarufomarginata; or a mixture thereof. Saccharification step (b) andfermentation step (c) may be carried out either sequentially orsimultaneously. A pullulanase and/or metalloprotease may be added duringsaccharification and/or fermentation when the process is carried out asa sequential saccharification and fermentation process and before orduring fermentation when steps (b) and (c) are carried outsimultaneously (SSF process). The pullulanase and/or metalloprotease mayalso advantageously be added before liquefaction (pre-liquefactiontreatment), i.e., before or during step (a), and/or after liquefaction(post liquefaction treatment), i.e., after step (a). The pullulanase ismost advantageously added before or during liquefaction, i.e., before orduring step (a). The fermentation product, such as especially ethanol,may optionally be recovered after fermentation, e.g., by distillation.The fermenting organism is preferably yeast, preferably a strain ofSaccharomyces cerevisiae. In a particular embodiment, the process of theinvention further comprises, prior to step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling (e.g., using a hammer mill);

y) forming a slurry comprising the starch-containing material and water.

In an embodiment the particle size is smaller than a #7 screen, e.g., a#6 screen. A #7 screen is usually used in conventional prior artprocesses. The aqueous slurry may contain from 10-55, e.g., 25-45 and30-40, w/w % dry solids (DS) of starch-containing material. The slurryis heated to above the gelatinization temperature and an alpha-amylasevariant may be added to initiate liquefaction (thinning). The slurry mayin an embodiment be jet-cooked to further gelatinize the slurry beforebeing subjected to alpha-amylase in step (a). Liquefaction may in anembodiment be carried out as a three-step hot slurry process. The slurryis heated to between 60-95° C., preferably between 70-90° C., such aspreferably between 80-85° C. at pH 4-6, preferably 4.5-5.5, andalpha-amylase variant, optionally together with a pullulanase and/orprotease, preferably metalloprotease, are added to initiate liquefaction(thinning). In an embodiment the slurry may then be jet-cooked at atemperature between 95-140° C., preferably 100-135° C., such as 105-125°C., for about 1-15 minutes, preferably for about 3-10 minutes,especially around about 5 minutes. The slurry is cooled to 60-95° C. andmore alpha-amylase variant and optionally pullulanase variant and/orprotease, preferably metalloprotease, is(are) added to finalizehydrolysis (secondary liquefaction). The liquefaction process is usuallycarried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5.Saccharification step (b) may be carried out using conditions well knownin the art. For instance, a full saccharification process may last up tofrom about 24 to about 72 hours, however, it is common only to do apre-saccharification of typically 40-90 minutes at a temperature between30-65° C., typically about 60° C., followed by complete saccharificationduring fermentation in a simultaneous saccharification and fermentationprocess (SSF process). Saccharification is typically carried out attemperatures from 20-75° C., preferably from 40-70° C., typically around60° C., and at a pH between 4 and 5, normally at about pH 4.5. The mostwidely used process to produce a fermentation product, especiallyethanol, is a simultaneous saccharification and fermentation (SSF)process, in which there is no holding stage for the saccharification,meaning that a fermenting organism, such as yeast, and enzyme(s), may beadded together. SSF may typically be carried out at a temperature from25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to34° C., preferably around about 32° C. In an embodiment fermentation isongoing for 6 to 120 hours, in particular 24 to 96 hours.

Beer Making

The alpha-amylase variants may also be used in a beer-making process andsimilar fermentations; the alpha-amylases will typically be added duringthe mashing process. The process is substantially similar to themilling, liquefaction, saccharification, and fermentation processesdescribed above.

Starch Slurry Processing with Stillage

Milled starch-containing material is combined with water and recycledthin-stillage resulting in an aqueous slurry. The slurry can comprisebetween 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to40%, 20 to 35% and 30-36% ds). In some embodiments, the recycledthin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to50%, 20 to 40% and also 20 to 30%).

Once the milled starch-containing material is combined with water andbackset, the pH is not adjusted in the slurry. Further the pH is notadjusted after the addition of a phytase and optionally an alpha-amylaseto the slurry. In an embodiment, the pH of the slurry will be in therange of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8, pH4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5and pH 5.2 to 5.9). The pH of the slurry may be between about pH 4.5 and5.2 depending on the amount of thin stillage added to the slurry and thetype of material comprising the thin stillage. For example, the pH ofthe thin stillage may be between pH 3.8 and pH 4.5.

During ethanol production, acids can be added to lower the pH in thebeer well, to reduce the risk of microbial contamination prior todistillation.

In some embodiments, a phytase is added to the slurry. In otherembodiments, in addition to phytase, an alpha-amylase is added to theslurry. In some embodiments, a phytase and alpha-amylase are added tothe slurry sequentially. In other embodiments, a phytase andalpha-amylase are added simultaneously. In some embodiments, the slurrycomprising a phytase and optionally, an alpha-amylase, are incubated(pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15minutes to 4 hours). In other embodiments, the slurry is incubated at atemperature in the range of about 40 to 115° C. (e.g., 45 to 80° C., 50to 70° C., 50 to 75° C., 60 to 110° C., 60 to 95° C., 70 to 110° C., 70to 85° C. and 77 to 86° C.).

In other embodiments, the slurry is incubated at a temperature of about0 to about 30° C. (e.g., 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10°C. and 0 to 5° C.) below the starch gelatinization temperature of thestarch-containing material. In some embodiments, the temperature isbelow about 68° C., below about 65° C., below about 62° C., below about60° C. and below about 55° C. In some embodiments, the temperature isabove about 45° C., above about 50° C., above about 55° C. and aboveabout 60° C. In some embodiments, the incubation of the slurrycomprising a phytase and an alpha-amylase at a temperature below thestarch gelatinization temperature is referred to as a primary (1°)liquefaction.

In one embodiment, the milled starch-containing material is corn ormilo. The slurry comprises 25 to 40% DS, the pH is in the range of 4.8to 5.2, and the slurry is incubated with a phytase and optionally analpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to75° C.

Currently, it is believed that commercially-available microbialalpha-amylases used in the liquefaction process are generally not stableenough to produce liquefied starch substrate from a dry mill processusing whole ground grain at a temperature above about 80° C. at a pHlevel that is less than pH 5.6. The stability of many commerciallyavailable alpha-amylases is reduced at a pH of less than about 4.0.

In a further liquefaction step, the incubated or pretreatedstarch-containing material is exposed to an increase in temperature suchas about 0 to about 45° C. above the starch gelatinization temperatureof the starch-containing material (e.g., 70° C. to 120° C., 70° C. to110° C., and 70° C. to 90° C.) for a period of time of about 2 minutesto about 6 hours (e.g., 2 minutes to 4 hrs, 90 minutes, 140 minutes and90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1hour to 2 hours. The temperature can be increased by a conventional hightemperature jet cooking system for a short period of time, for example,for 1 to 15 minutes. Then the starch maybe further hydrolyzed at atemperature ranging from about 75° C. to 95° C. (e.g., 80° C. to 90° C.and 80° C. to 85° C.) for a period of about 15 to 150 minutes (e.g., 30to 120 minutes). In a preferred embodiment, the pH is not adjustedduring these process steps and the pH of the liquefied mash is in therange of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, andpH 5.0 to 5.2). In some embodiments, a second dose of thermostablealpha-amylase is added to the secondary liquefaction step, but in otherembodiments there is no additional dosage of alpha-amylase.

The incubation and liquefaction steps may be followed bysaccharification and fermentation steps well known in the art.

Distillation

Optionally, following fermentation, an alcohol (e.g., ethanol) may beextracted by, for example, distillation and optionally followed by oneor more process steps.

In some embodiments, the yield of ethanol produced by the methodsprovided herein is at least 8%, at least 10%, at least 12%, at least14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) andat least 23% v/v. The ethanol obtained according to the process providedherein may be used as, for example, fuel ethanol, drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

By-Products

Left over from the fermentation is the grain, which is typically usedfor animal feed either in liquid or dried form. In further embodiments,the end product may include the fermentation co-products such asdistiller's dried grains (DDG) and distiller's dried grain plus solubles(DDGS), which may be used, for example, as an animal feed.

Further details on how to carry out liquefaction, saccharification,fermentation, distillation, and recovery of ethanol are well known tothe skilled person.

According to the process provided herein, the saccharification andfermentation may be carried out simultaneously or separately.

Pulp and Paper Production

The alpha-amylase variants may also be used in the production oflignocellulosic materials, such as pulp, paper and cardboard, fromstarch reinforced waste paper and cardboard, especially where re-pulpingoccurs at pH above 7 and where amylases facilitate the disintegration ofthe waste material through degradation of the reinforcing starch. Thealpha-amylase variants are especially useful in a process for producinga papermaking pulp from starch-coated printed-paper. The process may beperformed as described in WO 95/14807, comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after stepa), and

c) separating ink particles from the pulp after steps a) and b).

The alpha-amylase variants may also be useful in modifying starch whereenzymatically modified starch is used in papermaking together withalkaline fillers such as calcium carbonate, kaolin and clays. With thealpha-amylase variants it is possible to modify the starch in thepresence of the filler thus allowing for a simpler integrated process.

Desizing of Textiles, Fabrics and Garments

The alpha-amylase variants may also be very useful in textile, fabric orgarment desizing. In the textile processing industry, alpha-amylases aretraditionally used as auxiliaries in the desizing process to facilitatethe removal of starch-containing size, which has served as a protectivecoating on weft yarns during weaving. Complete removal of the sizecoating after weaving is important to ensure optimum results in thesubsequent processes, in which the fabric is scoured, bleached and dyed.Enzymatic starch breakdown is preferred because it does not involve anyharmful effect on the fiber material. In order to reduce processing costand increase mill throughput, the desizing process is sometimes combinedwith the scouring and bleaching steps. In such cases, non-enzymaticauxiliaries such as alkali or oxidation agents are typically used tobreak down the starch, because traditional alpha-amylases are not verycompatible with high pH levels and bleaching agents. The non-enzymaticbreakdown of the starch size leads to some fiber damage because of therather aggressive chemicals used. Accordingly, it would be desirable touse the alpha-amylase variants as they have an improved performance inalkaline solutions. The alpha-amylase variants may be used alone or incombination with a cellulase when desizing cellulose-containing fabricor textile.

Desizing and bleaching processes are well known in the art. Forinstance, such processes are described in e.g., WO 95/21247, U.S. Pat.No. 4,643,736, EP 119920, which are hereby incorporated by reference.

Cleaning Processes and Detergent Compositions

The alpha-amylase variants may be added as a component of a detergentcomposition for various cleaning or washing processes, including laundryand dishwashing. For example, the variants may be used in the detergentcompositions described in WO 96/23874 and WO 97/07202.

The alpha-amylase variants may be incorporated in detergents atconventionally employed concentrations. For example, a variant of theinvention may be incorporated in an amount corresponding to 0.00001-10mg (calculated as pure, active enzyme protein) of alpha-amylase perliter of wash/dishwash liquor using conventional dosing levels ofdetergent.

The detergent composition may for example be formulated as a hand ormachine laundry detergent composition, including a laundry additivecomposition suitable for pretreatment of stained fabrics and a rinseadded fabric softener composition or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

The detergent composition may further comprise one or more otherenzymes, such as a lipase, peroxidase, protease, another amylolyticenzyme, e.g., another alpha-amylase, glucoamylase, maltogenic amylase,CGTase, cellulase, mannanase (such as Mannaway™ from Novozymes,Denmark)), pectinase, pectin lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,e.g., a separate additive or a combined additive, can be formulated,e.g., granulate, a liquid, a slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols, fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition may be in any convenient form, e.g., a bar, atablet, a powder, a granule, a paste or a liquid. A liquid detergent maybe aqueous, typically containing up to about 70% water and 0 to about30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of fromabout 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0 to about 65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleiclacrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxy acids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and thecomposition may be formulated as described in, e.g., WO 92/19708 and WO92/19709.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilre-deposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

The detergent compositions may comprise any enzyme in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.055 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

One or more of the variant enzymes described herein may additionally beincorporated in the detergent formulations disclosed in WO 97/07202,which is hereby incorporated as reference.

This disclosure includes further detail in the following examples, whichare not in any way intended to limit the scope of what is claimed. Thefollowing examples are thus offered to illustrate, but not to limit whatis claimed.

EXAMPLES Assay for Determination of Residual Alpha-Amylase Activity

Residual alpha-amylase activity is determined by a method employing theEnzChek® substrate. The substrate in the EnzChek® Ultra Amylase AssayKit (E33651, Invitrogen, La Jolla, Calif., USA) is a corn starchderivative, DQ™ starch, which is corn starch labeled with BODIPY® FL dyeto such a degree that fluorescence is quenched.

One vial containing approx. 1 mg lyophilized substrate is dissolved in100 microliters of 50 mM sodium acetate (pH 4.0). The vial is vortexedfor 20 seconds and left at room temperature, in the dark, withoccasional mixing until dissolved. Then 900 microliters of 100 mMacetate, 0.01% (W/v) TRITON® X100, 0.12 mM CaCl₂, pH 5.5 is added,vortexed thoroughly and stored at room temperature, in the dark untilready to use. The substrate working solution is prepared by diluting10-fold in residual activity buffer (100 mM acetate, 0.01% (w/v) TRITON®X100, 0.12 mM CaCl₂, pH 5.5) giving a substrate concentration of 100micrograms/ml. Immediately after incubation the enzyme is diluted to aconcentration of 15 ng enzyme protein/mL in 100 mM acetate, 0.01% (W/v)TRITON® X100, 0.12 mM CaCl₂, pH 5.5.

For the assay, 25 microliters of the substrate working solution is mixedfor 10 second with 25 microliters of the diluted enzyme in a black 384well microtiter plate. The fluorescence intensity is measured(excitation: 485 nm, emission: 555 nm) once every minute for 15 minutesin each well at 25° C. and the V_(max) is calculated as the slope of theplot of fluorescence intensity against time. The plot should be linearand the residual activity assay has been adjusted so that the dilutedreference enzyme solution is within the linear range of the activityassay.

Glucoamylase Activity (AGU)

A Glucoamylase Unit (AGU) is defined as the amount of enzyme, whichhydrolyzes 1 micromole maltose per minute under the standard conditions37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M,reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37°C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

Example 1 Stability of Alpha-Amylase Variants

The stability of a reference alpha-amylase (Bacillus stearothermophilusalpha-amylase with the mutations I181*+G182*+N193F truncated to 491amino acids (SEQ ID NO: 6)) and alpha-amylase variants thereof wasdetermined by incubating the reference alpha-amylase and variants at pH4.5 and 5.5 and temperatures of 75° C. and 85° C. with 0.12 mM CaCl₂)followed by residual activity determination using the EnzChek® substrate(EnzChek® Ultra Amylase assay kit, E33651, Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 0.5and 1 or 5 and 10 ppm (micrograms/ml) in enzyme dilution buffer (10 mMacetate, 0.01% Triton X100, 0.12 mM CaCl₂, pH 5.0). Twenty microlitersenzyme sample was transferred to 48-well PCR MTP and 180 microlitersstability buffer (150 mM acetate, 150 mM MES, 0.01% Triton X100, 0.12 mMCaCl₂, pH 4.5 or 5.5) was added to each well and mixed. The assay wasperformed using two concentrations of enzyme in duplicates. Beforeincubation at 75° C. or 85° C., 20 microliters was withdrawn and storedon ice as control samples. Incubation was performed in a PCR machine at75° C. and 85° C.

After incubation samples were diluted to 15 ng/ml in residual activitybuffer (100 mM Acetate, 0.01% Triton X100, 0.12 mM CaCl₂, pH 5.5) and 25microliters diluted enzyme was transferred to black 384-MTP. Residualactivity was determined using the EnzChek substrate by adding 25microliters substrate solution (100 micrograms/ml) to each well.Fluorescence was determined every minute for 15 minutes using excitationfilter at 485-P nm and emission filter at 555 nm (fluorescence reader isPolarstar, BMG). The residual activity was normalized to control samplesfor each setup.

Assuming logarithmic decay half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined for thereference alpha-amylase and variant thereof as shown in Table 1.

TABLE 1 T½ (min) T½ (min) T½ (min) (pH 4.5, 75° C., (pH 4.5, 85° C., (pH5.5, 85° C., Mutations 0.12 mM CaCl₂) 0.12 mM CaCl₂) 0.12 mM CaCl₂)Reference amylase 21 4 111 Reference Alpha-Amylase with the 32 6 301substitution V59A Reference Alpha-Amylase with the >180 29 NDsubstitutions V59A + Q89R + E129V + D165N + K177L + R179E + H208Y +K220P + N224L + Q254S Reference Alpha-Amylase with the >180 23 NDsubstitutions V59A + Q89R + E129V + W166F + K177L + R179E + H208Y +K220P + N224L + Q254S Reference Alpha-Amylase with the >180 49 NDsubstitutions V59A + Q89R + E129V + K177L + R179E + H208Y + K220P +N224L + Q254S + M284V Reference Alpha-Amylase with the >180 78 NDsubstitutions V59A + E129V + D165N + K177L + R179E + H208Y + K220P +N224L + S242Q + Q254S Reference Alpha-Amylase with the >180 59 NDsubstitutions V59A + E129V + E168A + K177L + R179E + H208Y + K220P +N224L + S242Q + Q254S Reference Alpha-Amylase with the >180 25 NDsubstitutions V59A + E129V + E168A + K177L + R179E + H208Y + K220P +N224L + Q254S Reference Alpha-Amylase with the >180 72 ND substitutionsV59A + E129V + K171E + K177L + R179E + H208Y + K220P + N224L + S242Q +Q254S Reference Alpha-Amylase with the >180 81 ND substitutions V59A +E129V + K177L + R179E + A184S + H208Y + K220P + N224L + S242Q + Q254SReference Alpha-Amylase with the >180 141 ND substitutions V59A +E129V + K177L + R179E + H208Y + K220P + N224L + S242Q + Q254S + M284VReference Alpha-Amylase with the 28 5 230 substitution V59E ReferenceAlpha-Amylase with the 28 5 210 substitution V59I ReferenceAlpha-Amylase with the 30 6 250 substitution V59Q ReferenceAlpha-Amylase with the 41 7.1 286 substitution G108A ReferenceAlpha-Amylase with the 149 22 ND substitutions V59A + Q89R + G112D +E129V + K177L + R179E + K220P + N224L + Q254S Reference Alpha-Amylasewith the >180 28 ND substitutions V59A + Q89R + E129V + K177L + R179E +H208Y + K220P + N224L + Q254S Reference Alpha-Amylase with the 112 16 NDsubstitutions V59A + Q89R + E129V + K177L + R179E + K220P + N224L +Q254S + D269E + D281N Reference Alpha-Amylase with the 168 21 NDsubstitutions V59A + Q89R + E129V + K177L + R179E + K220P + N224L +Q254S + I270L Reference Alpha-Amylase with the >180 24 ND substitutionsV59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + H274KReference Alpha-Amylase with the 91 15 ND substitutions V59A + Q89R +E129V + K177L + R179E + K220P + N224L + Q254S + Y276F ReferenceAlpha-Amylase with the 141 41 ND substitutions V59A + E129V + R157Y +K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylasewith the >180 62 ND substitutions V59A + E129V + K177L + R179E + H208Y +K220P + N224L + S242Q + Q254S Reference Alpha-Amylase with the >18049 >480 substitutions V59A + E129V + K177L + R179E + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase with the >180 53 ND substitutionsV59A + E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274KReference Alpha-Amylase with the >180 57 ND substitutions V59A + E129V +K177L + R179E + K220P + N224L + S242Q + Q254S + Y276F ReferenceAlpha-Amylase with the >180 37 ND substitutions V59A + E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + D281N Reference Alpha-Amylasewith the >180 51 ND substitutions V59A + E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + M284T Reference Alpha-Amylase with the >180 45ND substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + G416V Reference Alpha-Amylase with the 143 21 >480 substitutionsV59A + E129V + K177L + R179E + K220P + N224L + Q254S ReferenceAlpha-Amylase with the >180 22 ND substitutions V59A + E129V + K177L +R179E + K220P + N224L + Q254S + M284T Reference Alpha-Amylase withthe >180 38 ND substitutions A91L + M96I + E129V + K177L + R179E +K220P + N224L + S242Q + Q254S Reference Alpha-Amylase with the 57 11 402substitutions E129V + K177L + R179E Reference Alpha-Amylase with the 17444 >480 substitutions E129V + K177L + R179E + K220P + N224L + S242Q +Q254S Reference Alpha-Amylase with the >180 49 >480 substitutionsE129V + K177L + R179E + K220P + N224L + S242Q + Q254S + Y276F + L427MReference Alpha-Amylase with the >180 49 >480 substitutions E129V +K177L + R179E + K220P + N224L + S242Q + Q254S + M284T ReferenceAlpha-Amylase with the 177 36 >480 substitutions E129V + K177L + R179E +K220P + N224L + S242Q + Q254S + N376* + I377* Reference Alpha-Amylasewith the 94 13 >480 substitutions E129V + K177L + R179E + K220P +N224L + Q254S Reference Alpha-Amylase with the 129 24 >480 substitutionsE129V + K177L + R179E + K220P + N224L + Q254S + M284T ReferenceAlpha-Amylase with the 148 30 >480 substitutions E129V + K177L + R179E +S242Q Reference Alpha-Amylase with the 78 9 >480 substitutions E129V +K177L + R179V Reference Alpha-Amylase with the 178 31 >480 substitutionsE129V + K177L + R179V + K220P + N224L + S242Q + Q254S ReferenceAlpha-Amylase with the 66 17 >480 substitutions K220P + N224L + S242Q +Q254S Reference Alpha-Amylase with the 30 6 159 substitutions K220P +N224L + Q254S Reference Alpha-Amylase with the 35 7 278 substitutionM284T Reference Alpha-Amylase with the 59 13 ND substitutions M284V NDnot determined

The results demonstrate that the alpha-amylase variants have asignificantly greater half-life and stability than the referencealpha-amylase.

Example 2 Production of Ethanol Using Alpha-Amylase Variants

Four small scale mashes of the reference alpha-amylase and twoalpha-amylase variants described in Example 1 were prepared as follows:about 54 g corn ground, about 51 g tap water, and about 45 g backsetwere mixed in a 250 mL plastic bottle to a total slurry weight of 150 g.The pH of the corn slurry was adjusted to 4.5. The enzymes were added tothe mashes at 2 micrograms of amylase per gram of dry solids. Forliquefaction, the alpha-amylases were added to the bottles and thebottles were mixed thoroughly and placed into a preheated 85° C. waterbath. The samples were held in the water bath for 2 hours at pH 4.5while being shaken every 10 minutes for the first 30 minutes and every30 minutes thereafter for the remainder of the 2 hour liquefaction. Thesamples were then cooled in an ice bath; pH was adjusted to 5.0, and0.75 mL urea and 0.45 mL penicillin were added to reach finalconcentrations of 1000 and 3 ppm in the mashes, respectively. Thesamples were then subjected to simultaneous saccharification andfermentation (SSF) with Sprizyme Fuel (a glucoamylase product sold byNovozymes).

Five gram aliquots of the mashes were transferred into pre-weighedconical centrifuge tubes, using 5 replicates per mash. SSF was thenperformed on these mashes in a 32° C. water bath for 54 hours usingSprizyme Fuel as the glucoamylase. The glucoamylase dose was 0.50 AGU/gDS for all fermentations. The CO₂ weight loss during SSF was measuredand ethanol was quantified using HPLC after 54 hours of SSF. The average54 hour HPLC SSF data are provided in Table 2 below.

TABLE 2 Ethanol Yields After 54 Hours Fermentation Ethanol,Alpha-Amylase g/L Std dev. Reference Alpha-Amylase 105.5946 0.3708Reference Alpha-Amylase with the substitutions 119.4197 0.8927 E129V +K177L + R179E Reference Alpha-Amylase with the substitutions 116.48670.5922 K220P + N224L + Q254S

The results demonstrate that the use of the alpha-amylase variantsresulted in a significantly greater yield of ethanol relative to thereference alpha-amylase.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention is further defined by the following paragraphs:

Paragraph 1. An isolated variant alpha-amylase, comprising asubstitution at three or more (several) positions corresponding to anyof positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171,177, 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276,281, 284, 416, and 427, wherein the variant has at least 65% and lessthan 100% sequence identity with the mature polypeptide of SEQ ID NO: 1,2, 3, 4, 5, 6, and/or 7 and the variant has alpha-amylase activity.Paragraph 2. The variant of paragraph 1, which comprises a substitutionat a position corresponding to position 59 with Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr,in particular with Ala, Gln, Glu, Gly, Ile, Leu, Pro, or Thr.Paragraph 3. The variant of paragraph 1 or 2, which comprises asubstitution at a position corresponding to position 89 with Ala, Arg,Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Arg, His, or Lys.Paragraph 4. The variant of any of paragraphs 1-3, which comprises asubstitution at a position corresponding to position 91 with Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ile, Leu, or Val.Paragraph 5. The variant of any of paragraphs 1-4, which comprises asubstitution at a position corresponding to position 96 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ile, Leu, or Val.Paragraph 6. The variant of any of paragraphs 1-5, which comprises asubstitution at a position corresponding to position 108 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ala.Paragraph 7. The variant of any of paragraphs 1-6, which comprises asubstitution at a position corresponding to position 112 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ala, Asp, or Glu.Paragraph 8. The variant of any of paragraphs 1-7, which comprises asubstitution at a position corresponding to position 129 with Ala, Arg,Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ala, Thr, or Val.Paragraph 9. The variant of any of paragraphs 1-8, which comprises asubstitution at a position corresponding to position 157 with Ala, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with His, Lys, Phe, or Tyr.Paragraph 10. The variant of any of paragraphs 1-9, which comprises asubstitution at a position corresponding to position 165 Ala, Arg, Asn,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val, in particular with Asn.Paragraph 11. The variant of any of paragraphs 1-10, which comprises asubstitution at a position corresponding to position 166 Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Tyr, or Val, in particular with Phe.Paragraph 12. The variant of any of paragraphs 1-11, which comprises asubstitution at a position corresponding to position 168 with Ala, Arg,Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ala.Paragraph 13. The variant of any of paragraphs 1-12, which comprises asubstitution at a position corresponding to position 171 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Glu.Paragraph 14. The variant of any of paragraphs 1-13, which comprises asubstitution at a position corresponding to position 177 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Arg, Leu, or Met.Paragraph 15. The variant of any of paragraphs 1-14, which comprises asubstitution at a position corresponding to position 179 with Ala, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Gln, Glu, Ile, Leu, Lys, or Val.Paragraph 16. The variant of any of paragraphs 1-15, which comprises asubstitution at a position corresponding to position 180 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Glu or Val.Paragraph 17. The variant of any of paragraphs 1-16, which comprises asubstitution at a position corresponding to position 181 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Glu, Gly, or Val.Paragraph 18. The variant of any of paragraphs 1-17, which comprises asubstitution at a position corresponding to position 184 with Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ser.Paragraph 19. The variant of any of paragraphs 1-18, which comprises asubstitution at a position corresponding to position 208 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Phe or Tyr.Paragraph 20. The variant of any of paragraphs 1-19, which comprises asubstitution at a position corresponding to position 220 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Pro.Paragraph 21. The variant of any of paragraphs 1-20, which comprises asubstitution at a position corresponding to position 224 with Ala, Arg,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Leu.Paragraph 22. The variant of any of paragraphs 1-21, which comprises asubstitution at a position corresponding to position 242 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr,Trp, Tyr, or Val, in particular with Ala, Asp, Glu, Gln, or Met.Paragraph 23. The variant of any of paragraphs 1-22, which comprises asubstitution at a position corresponding to position 254 with Ala, Arg,Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ala, Ser, or Thr.Paragraph 24. The variant of any of paragraphs 1-23, which comprises asubstitution at a position corresponding to position 269 with Ala, Arg,Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Gln or Glu.Paragraph 25. The variant of any of paragraphs 1-24, which comprises asubstitution at a position corresponding to position 270 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Leu, Thr, or Val.Paragraph 26. The variant of any of paragraphs 1-25, which comprises asubstitution at a position corresponding to position 274 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Arg, Gln, Glu, Lys, or Phe.Paragraph 27. The variant of any of paragraphs 1-26, which comprises asubstitution at a position corresponding to position 276 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, or Val, in particular with Phe.Paragraph 28. The variant of any of paragraphs 1-27, which comprises asubstitution at a position corresponding to position 281 with Ala, Arg,Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Asn or Ser.Paragraph 29. The variant of any of paragraphs 1-28, which comprises asubstitution at a position corresponding to position 284 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with His, Thr, or Val.Paragraph 30. The variant of any of paragraphs 1-29, which comprises asubstitution at a position corresponding to position 416 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ala, Asn, Asp, Ser, Thr, or Val.Paragraph 31. The variant of any of paragraphs 1-30, which comprises asubstitution at a position corresponding to position 427 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ile, Met, or Val.Paragraph 32. The variant of any of paragraphs 1-31, comprising asubstitution at three positions corresponding to any of positions 59,89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181,184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and427, in particular, at the positions corresponding to positions 129,177, and 179 or positions 220, 242, and 254.Paragraph 33. The variant of paragraph 32, wherein the three positionsare selected from the group consisting of:

Positions 59, 89, and 129;

Positions 59, 89, and 177;

Positions 59, 89, and 179;

Positions 59, 89, and 180;

Positions 59, 89, and 181;

Positions 59, 89, and 220;

Positions 59, 89, and 224;

Positions 59, 89, and 242;

Positions 59, 89, and 254;

Positions 59, 108, and 284;

Positions 59, 129, and 177;

Positions 59, 129, and 179;

Positions 59, 129, and 180;

Positions 59, 129, and 181;

Positions 59, 129, and 220;

Positions 59, 129, and 224;

Positions 59, 129, and 242;

Positions 59, 129, and 254;

Positions 59, 177, and 179;

Positions 59, 177, and 180;

Positions 59, 177, and 181;

Positions 59, 177, and 220;

Positions 59, 177, and 224;

Positions 59, 177, and 242;

Positions 59, 177, and 254;

Positions 59, 179, and 181;

Positions 59, 179, and 220;

Positions 59, 179, and 224;

Positions 59, 179, and 242;

Positions 59, 179, and 254;

Positions 59, 180, and 181;

Positions 59, 180, and 220;

Positions 59, 180, and 224;

Positions 59, 180, and 242;

Positions 59, 180, and 254;

Positions 59, 181, and 220;

Positions 59, 181, and 224;

Positions 59, 181, and 242;

Positions 59, 181, and 254;

Positions 59, 220, and 224;

Positions 59, 220, and 242;

Positions 59, 220, and 254;

Positions 59, 224, and 242;

Positions 59, 224, and 254;

Positions 59, 242, and 254;

Positions 89, 129, and 177;

Positions 89, 129, and 179;

Positions 89, 129, and 180;

Positions 89, 129, and 181;

Positions 89, 129, and 220;

Positions 89, 129, and 224;

Positions 89, 129, and 242;

Positions 89, 129, and 254;

Positions 89, 177, and 179;

Positions 89, 177, and 180;

Positions 89, 177, and 181;

Positions 89, 177, and 220;

Positions 89, 177, and 224;

Positions 89, 177, and 242;

Positions 89, 177, and 254;

Positions 89, 179, and 181;

Positions 89, 179, and 220;

Positions 89, 179, and 224;

Positions 89, 179, and 242;

Positions 89, 179, and 254;

Positions 89, 180, and 181;

Positions 89, 180, and 220;

Positions 89, 180, and 224;

Positions 89, 180, and 242;

Positions 89, 180, and 254;

Positions 89, 181, and 220;

Positions 89, 181, and 224;

Positions 89, 181, and 242;

Positions 89, 181, and 254;

Positions 89, 220, and 224;

Positions 89, 220, and 242;

Positions 89, 220, and 254;

Positions 89, 224, and 242;

Positions 89, 224, and 254;

Positions 89, 242, and 254;

Positions 129, 177, and 179;

Positions 129, 177, and 180;

Positions 129, 177, and 181;

Positions 129, 177, and 220;

Positions 129, 177, and 224;

Positions 129, 177, and 242;

Positions 129, 177, and 254;

Positions 129, 179, and 181;

Positions 129, 179, and 220;

Positions 129, 179, and 224;

Positions 129, 179, and 242;

Positions 129, 179, and 254;

Positions 129, 180, and 181;

Positions 129, 180, and 220;

Positions 129, 180, and 224;

Positions 129, 180, and 242;

Positions 129, 180, and 254;

Positions 129, 181, and 220;

Positions 129, 181, and 224;

Positions 129, 181, and 242;

Positions 129, 181, and 254;

Positions 129, 220, and 224;

Positions 129, 220, and 242;

Positions 129, 220, and 254;

Positions 129, 224, and 242;

Positions 129, 224, and 254;

Positions 129, 242, and 254;

Positions 177, 179, and 181;

Positions 177, 179, and 220;

Positions 177, 179, and 224;

Positions 177, 179, and 242;

Positions 177, 179, and 254;

Positions 177, 180, and 181;

Positions 177, 180, and 220;

Positions 177, 180, and 224;

Positions 177, 180, and 242;

Positions 177, 180, and 254;

Positions 177, 181, and 220;

Positions 177, 181, and 224;

Positions 177, 181, and 242;

Positions 177, 181, and 254;

Positions 177, 220, and 224;

Positions 177, 220, and 242;

Positions 177, 220, and 254;

Positions 177, 224, and 242;

Positions 177, 224, and 254;

Positions 177, 242, and 254;

Positions 179, 181, and 220;

Positions 179, 181, and 224;

Positions 179, 181, and 242;

Positions 179, 181, and 254;

Positions 179, 220, and 224;

Positions 179, 220, and 242;

Positions 179, 220, and 254;

Positions 179, 224, and 242;

Positions 179, 224, and 254;

Positions 179, 242, and 254;

Positions 180, 181, and 220;

Positions 180, 181, and 224;

Positions 180, 181, and 242;

Positions 180, 181, and 254;

Positions 180, 220, and 224;

Positions 180, 220, and 242;

Positions 180, 220, and 254;

Positions 180, 224, and 242;

Positions 180, 224, and 254;

Positions 180, 242, and 254;

Positions 181, 220, and 224;

Positions 181, 220, and 242;

Positions 181, 220, and 254;

Positions 181, 224, and 242;

Positions 181, 224, and 254;

Positions 181, 242, and 254;

Positions 220, 224, and 242;

Positions 220, 224, and 254; and

Positions 224, 242, and 254.

Paragraph 34. The variant of any of paragraphs 1-31, comprising asubstitution at four positions corresponding to any of positions 59, 89,91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184,208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and 427, inparticular, at the positions corresponding to positions 129, 177, 179,and 208; positions 129, 177, 179, and 242; positions 129, 177, 179, and284; positions 208, 220, 224, and 254; positions 220, 224, 242, and 254;or positions 220, 224, 254, and 284.Paragraph 35. The variant of paragraph 34, wherein the four positionsare selected from the group consisting of:

Positions 59, 89, 129, and 177;

Positions 59, 89, 129, and 179;

Positions 59, 89, 129, and 180;

Positions 59, 89, 129, and 181;

Positions 59, 89, 129, and 220;

Positions 59, 89, 129, and 224;

Positions 59, 89, 129, and 242;

Positions 59, 89, 129, and 254;

Positions 59, 89, 177, and 179;

Positions 59, 89, 177, and 180;

Positions 59, 89, 177, and 181;

Positions 59, 89, 177, and 220;

Positions 59, 89, 177, and 224;

Positions 59, 89, 177, and 242;

Positions 59, 89, 177, and 254;

Positions 59, 89, 179, and 181;

Positions 59, 89, 179, and 220;

Positions 59, 89, 179, and 224;

Positions 59, 89, 179, and 242;

Positions 59, 89, 179, and 254;

Positions 59, 89, 180, and 181;

Positions 59, 89, 180, and 220;

Positions 59, 89, 180, and 224;

Positions 59, 89, 180, and 242;

Positions 59, 89, 180, and 254;

Positions 59, 89, 181, and 220;

Positions 59, 89, 181, and 224;

Positions 59, 89, 181, and 242;

Positions 59, 89, 181, and 254;

Positions 59, 89, 220, and 224;

Positions 59, 89, 220, and 242;

Positions 59, 89, 220, and 254;

Positions 59, 89, 224, and 242;

Positions 59, 89, 224, and 254;

Positions 59, 89, 242, and 254;

Positions 59, 108, 242, and 284;

Positions 59, 129, 177, and 179;

Positions 59, 129, 177, and 180;

Positions 59, 129, 177, and 181;

Positions 59, 129, 177, and 220;

Positions 59, 129, 177, and 224;

Positions 59, 129, 177, and 242;

Positions 59, 129, 177, and 254;

Positions 59, 129, 179, and 181;

Positions 59, 129, 179, and 220;

Positions 59, 129, 179, and 224;

Positions 59, 129, 179, and 242;

Positions 59, 129, 179, and 254;

Positions 59, 129, 180, and 181;

Positions 59, 129, 180, and 220;

Positions 59, 129, 180, and 224;

Positions 59, 129, 180, and 242;

Positions 59, 129, 180, and 254;

Positions 59, 129, 181, and 220;

Positions 59, 129, 181, and 224;

Positions 59, 129, 181, and 242;

Positions 59, 129, 181, and 254;

Positions 59, 129, 220, and 224;

Positions 59, 129, 220, and 242;

Positions 59, 129, 220, and 254;

Positions 59, 129, 224, and 242;

Positions 59, 129, 224, and 254;

Positions 59, 129, 242, and 254;

Positions 59, 177, 179, and 181;

Positions 59, 177, 179, and 220;

Positions 59, 177, 179, and 224;

Positions 59, 177, 179, and 242;

Positions 59, 177, 179, and 254;

Positions 59, 177, 180, and 181;

Positions 59, 177, 180, and 220;

Positions 59, 177, 180, and 224;

Positions 59, 177, 180, and 242;

Positions 59, 177, 180, and 254;

Positions 59, 177, 181, and 220;

Positions 59, 177, 181, and 224;

Positions 59, 177, 181, and 242;

Positions 59, 177, 181, and 254;

Positions 59, 177, 220, and 224;

Positions 59, 177, 220, and 242;

Positions 59, 177, 220, and 254;

Positions 59, 177, 224, and 242;

Positions 59, 177, 224, and 254;

Positions 59, 177, 242, and 254;

Positions 59, 179, 181, and 220;

Positions 59, 179, 181, and 224;

Positions 59, 179, 181, and 242;

Positions 59, 179, 181, and 254;

Positions 59, 179, 220, and 224;

Positions 59, 179, 220, and 242;

Positions 59, 179, 220, and 254;

Positions 59, 179, 224, and 242;

Positions 59, 179, 224, and 254;

Positions 59, 179, 242, and 254;

Positions 59, 180, 181, and 220;

Positions 59, 180, 181, and 224;

Positions 59, 180, 181, and 242;

Positions 59, 180, 181, and 254;

Positions 59, 180, 220, and 224;

Positions 59, 180, 220, and 242;

Positions 59, 180, 220, and 254;

Positions 59, 180, 224, and 242;

Positions 59, 180, 224, and 254;

Positions 59, 180, 242, and 254;

Positions 59, 181, 220, and 224;

Positions 59, 181, 220, and 242;

Positions 59, 181, 220, and 254;

Positions 59, 181, 224, and 242;

Positions 59, 181, 224, and 254;

Positions 59, 181, 242, and 254;

Positions 59, 220, 224, and 242;

Positions 59, 220, 224, and 254;

Positions 59, 220, 242, and 254;

Positions 59, 224, 242, and 254;

Positions 89, 129, 177, and 179;

Positions 89, 129, 177, and 180;

Positions 89, 129, 177, and 181;

Positions 89, 129, 177, and 220;

Positions 89, 129, 177, and 224;

Positions 89, 129, 177, and 242;

Positions 89, 129, 177, and 254;

Positions 89, 129, 179, and 181;

Positions 89, 129, 179, and 220;

Positions 89, 129, 179, and 224;

Positions 89, 129, 179, and 242;

Positions 89, 129, 179, and 254;

Positions 89, 129, 180, and 181;

Positions 89, 129, 180, and 220;

Positions 89, 129, 180, and 224;

Positions 89, 129, 180, and 242;

Positions 89, 129, 180, and 254;

Positions 89, 129, 181, and 220;

Positions 89, 129, 181, and 224;

Positions 89, 129, 181, and 242;

Positions 89, 129, 181, and 254;

Positions 89, 129, 220, and 224;

Positions 89, 129, 220, and 242;

Positions 89, 129, 220, and 254;

Positions 89, 129, 224, and 242;

Positions 89, 129, 224, and 254;

Positions 89, 129, 242, and 254;

Positions 89, 177, 179, and 181;

Positions 89, 177, 179, and 220;

Positions 89, 177, 179, and 224;

Positions 89, 177, 179, and 242;

Positions 89, 177, 179, and 254;

Positions 89, 177, 180, and 181;

Positions 89, 177, 180, and 220;

Positions 89, 177, 180, and 224;

Positions 89, 177, 180, and 242;

Positions 89, 177, 180, and 254;

Positions 89, 177, 181, and 220;

Positions 89, 177, 181, and 224;

Positions 89, 177, 181, and 242;

Positions 89, 177, 181, and 254;

Positions 89, 179, 181, and 220;

Positions 89, 179, 181, and 224;

Positions 89, 179, 181, and 242;

Positions 89, 179, 181, and 254;

Positions 89, 179, 220, and 224;

Positions 89, 179, 220, and 242;

Positions 89, 179, 220, and 254;

Positions 89, 179, 224, and 242;

Positions 89, 179, 224, and 254;

Positions 89, 179, 242, and 254;

Positions 89, 180, 181, and 220;

Positions 89, 180, 181, and 224;

Positions 89, 180, 181, and 242;

Positions 89, 180, 181, and 254;

Positions 89, 180, 220, and 224;

Positions 89, 180, 220, and 242;

Positions 89, 180, 220, and 254;

Positions 89, 180, 224, and 242;

Positions 89, 180, 224, and 254;

Positions 89, 180, 242, and 254;

Positions 89, 181, 220, and 224;

Positions 89, 181, 220, and 242;

Positions 89, 181, 220, and 254;

Positions 89, 181, 224, and 242;

Positions 89, 181, 224, and 254;

Positions 89, 181, 242, and 254;

Positions 89, 220, 224, and 242;

Positions 89, 220, 224, and 254;

Positions 89, 220, 242, and 254;

Positions 89, 224, 242, and 254;

Positions 129, 177, 179, and 181;

Positions 129, 177, 179, and 220;

Positions 129, 177, 179, and 224;

Positions 129, 177, 179, and 242;

Positions 129, 177, 179, and 254;

Positions 129, 177, 179, and 284;

Positions 129, 177, 180, and 181;

Positions 129, 177, 180, and 220;

Positions 129, 177, 180, and 224;

Positions 129, 177, 180, and 242;

Positions 129, 177, 180, and 254;

Positions 129, 177, 181, and 220;

Positions 129, 177, 181, and 224;

Positions 129, 177, 181, and 242;

Positions 129, 177, 181, and 254;

Positions 129, 177, 220, and 224;

Positions 129, 177, 220, and 242;

Positions 129, 177, 220, and 254;

Positions 129, 177, 224, and 242;

Positions 129, 177, 224, and 254;

Positions 129, 177, 242, and 254;

Positions 129, 179, 181, and 220;

Positions 129, 179, 181, and 224;

Positions 129, 179, 181, and 242;

Positions 129, 179, 181, and 254;

Positions 129, 179, 220, and 224;

Positions 129, 179, 220, and 242;

Positions 129, 179, 220, and 254;

Positions 129, 179, 224, and 242;

Positions 129, 179, 224, and 254;

Positions 129, 179, 242, and 254;

Positions 129, 180, 181, and 220;

Positions 129, 180, 181, and 224;

Positions 129, 180, 181, and 242;

Positions 129, 180, 181, and 254;

Positions 129, 180, 220, and 224;

Positions 129, 180, 220, and 242;

Positions 129, 180, 220, and 254;

Positions 129, 180, 224, and 242;

Positions 129, 180, 224, and 254;

Positions 129, 180, 242, and 254;

Positions 129, 181, 220, and 224;

Positions 129, 181, 220, and 242;

Positions 129, 181, 220, and 254;

Positions 129, 181, 224, and 242;

Positions 129, 181, 224, and 254;

Positions 129, 181, 242, and 254;

Positions 129, 220, 224, and 242;

Positions 129, 220, 224, and 254;

Positions 129, 220, 242, and 254;

Positions 129, 224, 242, and 254;

Positions 177, 179, 181, and 220;

Positions 177, 179, 181, and 224;

Positions 177, 179, 181, and 242;

Positions 177, 179, 181, and 254;

Positions 177, 179, 220, and 224;

Positions 177, 179, 220, and 242;

Positions 177, 179, 220, and 254;

Positions 177, 179, 224, and 242;

Positions 177, 179, 224, and 254;

Positions 177, 179, 242, and 254;

Positions 177, 180, 181, and 220;

Positions 177, 180, 181, and 224;

Positions 177, 180, 181, and 242;

Positions 177, 180, 181, and 254;

Positions 177, 180, 220, and 224;

Positions 177, 180, 220, and 242;

Positions 177, 180, 220, and 254;

Positions 177, 180, 224, and 242;

Positions 177, 180, 224, and 254;

Positions 177, 180, 242, and 254;

Positions 177, 181, 220, and 224;

Positions 177, 181, 220, and 242;

Positions 177, 181, 220, and 254;

Positions 177, 181, 224, and 242;

Positions 177, 181, 224, and 254;

Positions 177, 181, 242, and 254;

Positions 177, 220, 224, and 242;

Positions 177, 220, 224, and 254;

Positions 177, 220, 242, and 254;

Positions 177, 224, 242, and 254;

Positions 179, 181, 220, and 224;

Positions 179, 181, 220, and 242;

Positions 179, 181, 220, and 254;

Positions 179, 181, 224, and 242;

Positions 179, 181, 224, and 254;

Positions 179, 181, 242, and 254;

Positions 179, 220, 224, and 242;

Positions 179, 220, 224, and 254;

Positions 179, 220, 242, and 254;

Positions 179, 224, 242, and 254;

Positions 180, 181, 220, and 224;

Positions 180, 181, 220, and 242;

Positions 180, 181, 220, and 254;

Positions 180, 181, 224, and 242;

Positions 180, 181, 224, and 254;

Positions 180, 181, 242, and 254;

Positions 180, 220, 224, and 242;

Positions 180, 220, 224, and 254;

Positions 180, 220, 242, and 254;

Positions 180, 224, 242, and 254;

Positions 181, 220, 224, and 242;

Positions 181, 220, 224, and 254;

Positions 181, 220, 242, and 254;

Positions 181, 224, 242, and 254; and

Positions 220, 224, 242, and 254.

Paragraph 36. The variant of any of paragraphs 1-31, comprising asubstitution at five positions corresponding to any of positions 59, 89,91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184,208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and 427, inparticular, at the positions corresponding to positions 129, 177, 179,208, and 242; positions 129, 177, 179, 208, and 284; positions 208, 220,224, 242, and 254; or positions 208, 220, 224, 254, and 284.Paragraph 37. The variant of paragraph 36, wherein the five positionsare selected from the group consisting of:

Positions 59, 89, 129, 177, and 179;

Positions 59, 89, 129, 177, and 180;

Positions 59, 89, 129, 177, and 181;

Positions 59, 89, 129, 177, and 220;

Positions 59, 89, 129, 177, and 224;

Positions 59, 89, 129, 177, and 242;

Positions 59, 89, 129, 177, and 254;

Positions 59, 89, 129, 179, and 181;

Positions 59, 89, 129, 179, and 220;

Positions 59, 89, 129, 179, and 224;

Positions 59, 89, 129, 179, and 242;

Positions 59, 89, 129, 179, and 254;

Positions 59, 89, 129, 180, and 181;

Positions 59, 89, 129, 180, and 220;

Positions 59, 89, 129, 180, and 224;

Positions 59, 89, 129, 180, and 242;

Positions 59, 89, 129, 180, and 254;

Positions 59, 89, 129, 181, and 220;

Positions 59, 89, 129, 181, and 224;

Positions 59, 89, 129, 181, and 242;

Positions 59, 89, 129, 181, and 254;

Positions 59, 89, 129, 220, and 224;

Positions 59, 89, 129, 220, and 242;

Positions 59, 89, 129, 220, and 254;

Positions 59, 89, 129, 224, and 242;

Positions 59, 89, 129, 224, and 254;

Positions 59, 89, 129, 242, and 254;

Positions 59, 89, 177, 179, and 181;

Positions 59, 89, 177, 179, and 220;

Positions 59, 89, 177, 179, and 224;

Positions 59, 89, 177, 179, and 242;

Positions 59, 89, 177, 179, and 254;

Positions 59, 89, 177, 180, and 181;

Positions 59, 89, 177, 180, and 220;

Positions 59, 89, 177, 180, and 224;

Positions 59, 89, 177, 180, and 242;

Positions 59, 89, 177, 180, and 254;

Positions 59, 89, 177, 181, and 220;

Positions 59, 89, 177, 181, and 224;

Positions 59, 89, 177, 181, and 242;

Positions 59, 89, 177, 181, and 254;

Positions 59, 89, 177, 220, and 224;

Positions 59, 89, 177, 220, and 242;

Positions 59, 89, 177, 220, and 254;

Positions 59, 89, 177, 224, and 242;

Positions 59, 89, 177, 224, and 254;

Positions 59, 89, 177, 242, and 254;

Positions 59, 89, 179, 181, and 220;

Positions 59, 89, 179, 181, and 224;

Positions 59, 89, 179, 181, and 242;

Positions 59, 89, 179, 181, and 254;

Positions 59, 89, 179, 220, and 224;

Positions 59, 89, 179, 220, and 242;

Positions 59, 89, 179, 220, and 254;

Positions 59, 89, 179, 224, and 242;

Positions 59, 89, 179, 224, and 254;

Positions 59, 89, 179, 242, and 254;

Positions 59, 89, 180, 181, and 220;

Positions 59, 89, 180, 181, and 224;

Positions 59, 89, 180, 181, and 242;

Positions 59, 89, 180, 181, and 254;

Positions 59, 89, 180, 220, and 224;

Positions 59, 89, 180, 220, and 242;

Positions 59, 89, 180, 220, and 254;

Positions 59, 89, 180, 224, and 242;

Positions 59, 89, 180, 224, and 254;

Positions 59, 89, 180, 242, and 254;

Positions 59, 89, 181, 220, and 224;

Positions 59, 89, 181, 220, and 242;

Positions 59, 89, 181, 220, and 254;

Positions 59, 89, 181, 224, and 242;

Positions 59, 89, 181, 224, and 254;

Positions 59, 89, 181, 242, and 254;

Positions 59, 89, 220, 224, and 242;

Positions 59, 89, 220, 224, and 254;

Positions 59, 89, 220, 242, and 254;

Positions 59, 89, 224, 242, and 254;

Positions 59, 129, 177, 179, and 181;

Positions 59, 129, 177, 179, and 220;

Positions 59, 129, 177, 179, and 224;

Positions 59, 129, 177, 179, and 242;

Positions 59, 129, 177, 179, and 254;

Positions 59, 129, 177, 180, and 181;

Positions 59, 129, 177, 180, and 220;

Positions 59, 129, 177, 180, and 224;

Positions 59, 129, 177, 180, and 242;

Positions 59, 129, 177, 180, and 254;

Positions 59, 129, 177, 181, and 220;

Positions 59, 129, 177, 181, and 224;

Positions 59, 129, 177, 181, and 242;

Positions 59, 129, 177, 181, and 254;

Positions 59, 129, 177, 220, and 224;

Positions 59, 129, 177, 220, and 242;

Positions 59, 129, 177, 220, and 254;

Positions 59, 129, 177, 224, and 242;

Positions 59, 129, 177, 224, and 254;

Positions 59, 129, 177, 242, and 254;

Positions 59, 129, 179, 181, and 220;

Positions 59, 129, 179, 181, and 224;

Positions 59, 129, 179, 181, and 242;

Positions 59, 129, 179, 181, and 254;

Positions 59, 129, 179, 220, and 224;

Positions 59, 129, 179, 220, and 242;

Positions 59, 129, 179, 220, and 254;

Positions 59, 129, 179, 224, and 242;

Positions 59, 129, 179, 224, and 254;

Positions 59, 129, 179, 242, and 254;

Positions 59, 129, 180, 181, and 220;

Positions 59, 129, 180, 181, and 224;

Positions 59, 129, 180, 181, and 242;

Positions 59, 129, 180, 181, and 254;

Positions 59, 129, 180, 220, and 224;

Positions 59, 129, 180, 220, and 242;

Positions 59, 129, 180, 220, and 254;

Positions 59, 129, 180, 224, and 242;

Positions 59, 129, 180, 224, and 254;

Positions 59, 129, 180, 242, and 254;

Positions 59, 129, 181, 220, and 224;

Positions 59, 129, 181, 220, and 242;

Positions 59, 129, 181, 220, and 254;

Positions 59, 129, 181, 224, and 242;

Positions 59, 129, 181, 224, and 254;

Positions 59, 129, 181, 242, and 254;

Positions 59, 129, 220, 224, and 242;

Positions 59, 129, 220, 224, and 254;

Positions 59, 129, 224, 242, and 254;

Positions 59, 177, 179, 181, and 220;

Positions 59, 177, 179, 181, and 224;

Positions 59, 177, 179, 181, and 242;

Positions 59, 177, 179, 181, and 254;

Positions 59, 177, 179, 220, and 224;

Positions 59, 177, 179, 220, and 242;

Positions 59, 177, 179, 220, and 254;

Positions 59, 177, 179, 224, and 242;

Positions 59, 177, 179, 224, and 254;

Positions 59, 177, 179, 242, and 254;

Positions 59, 177, 180, 181, and 220;

Positions 59, 177, 180, 181, and 224;

Positions 59, 177, 180, 181, and 242;

Positions 59, 177, 180, 181, and 254;

Positions 59, 177, 180, 220, and 224;

Positions 59, 177, 180, 220, and 242;

Positions 59, 177, 180, 220, and 254;

Positions 59, 177, 180, 224, and 242;

Positions 59, 177, 180, 224, and 254;

Positions 59, 177, 180, 242, and 254;

Positions 59, 177, 181, 220, and 224;

Positions 59, 177, 181, 220, and 242;

Positions 59, 177, 181, 220, and 254;

Positions 59, 177, 181, 224, and 242;

Positions 59, 177, 181, 224, and 254;

Positions 59, 177, 181, 242, and 254;

Positions 59, 177, 220, 224, and 242;

Positions 59, 177, 220, 224, and 254;

Positions 59, 177, 224, 242, and 254;

Positions 59, 179, 181, 220, and 224;

Positions 59, 179, 181, 220, and 242;

Positions 59, 179, 181, 220, and 254;

Positions 59, 179, 181, 224, and 242;

Positions 59, 179, 181, 224, and 254;

Positions 59, 179, 181, 242, and 254;

Positions 59, 179, 220, 224, and 242;

Positions 59, 179, 220, 224, and 254;

Positions 59, 179, 220, 242, and 254;

Positions 59, 180, 181, 220, and 224;

Positions 59, 180, 181, 220, and 242;

Positions 59, 180, 181, 220, and 254;

Positions 59, 180, 181, 224, and 242;

Positions 59, 180, 181, 224, and 254;

Positions 59, 180, 181, 242, and 254;

Positions 59, 180, 220, 224, and 242;

Positions 59, 180, 220, 224, and 254;

Positions 59, 180, 220, 242, and 254;

Positions 59, 181, 220, 224, and 242;

Positions 59, 181, 220, 224, and 254;

Positions 59, 181, 220, 242, and 254;

Positions 59, 220, 224, 242, and 254;

Positions 89, 129, 177, 179, and 181;

Positions 89, 129, 177, 179, and 220;

Positions 89, 129, 177, 179, and 224;

Positions 89, 129, 177, 179, and 242;

Positions 89, 129, 177, 179, and 254;

Positions 89, 129, 177, 180, and 181;

Positions 89, 129, 177, 180, and 220;

Positions 89, 129, 177, 180, and 224;

Positions 89, 129, 177, 180, and 242;

Positions 89, 129, 177, 180, and 254;

Positions 89, 129, 177, 181, and 220;

Positions 89, 129, 177, 181, and 224;

Positions 89, 129, 177, 181, and 242;

Positions 89, 129, 177, 181, and 254;

Positions 89, 129, 177, 220, and 224;

Positions 89, 129, 177, 220, and 242;

Positions 89, 129, 177, 220, and 254;

Positions 89, 129, 177, 224, and 242;

Positions 89, 129, 177, 224, and 254;

Positions 89, 129, 177, 242, and 254;

Positions 89, 129, 179, 181, and 220;

Positions 89, 129, 179, 181, and 224;

Positions 89, 129, 179, 181, and 242;

Positions 89, 129, 179, 181, and 254;

Positions 89, 129, 179, 220, and 224;

Positions 89, 129, 179, 220, and 242;

Positions 89, 129, 179, 220, and 254;

Positions 89, 129, 179, 224, and 242;

Positions 89, 129, 179, 224, and 254;

Positions 89, 129, 179, 242, and 254;

Positions 89, 129, 180, 181, and 220;

Positions 89, 129, 180, 181, and 224;

Positions 89, 129, 180, 181, and 242;

Positions 89, 129, 180, 181, and 254;

Positions 89, 129, 180, 220, and 224;

Positions 89, 129, 180, 220, and 242;

Positions 89, 129, 180, 220, and 254;

Positions 89, 129, 180, 224, and 242;

Positions 89, 129, 180, 224, and 254;

Positions 89, 129, 180, 242, and 254;

Positions 89, 129, 181, 220, and 224;

Positions 89, 129, 181, 220, and 242;

Positions 89, 129, 181, 220, and 254;

Positions 89, 129, 181, 224, and 242;

Positions 89, 129, 181, 224, and 254;

Positions 89, 129, 180, 242, and 254;

Positions 89, 177, 179, 181, and 220;

Positions 89, 177, 179, 181, and 224;

Positions 89, 177, 179, 181, and 242;

Positions 89, 177, 179, 181, and 254;

Positions 89, 177, 179, 220, and 224;

Positions 89, 177, 179, 220, and 242;

Positions 89, 177, 179, 220, and 254;

Positions 89, 177, 179, 224, and 242;

Positions 89, 177, 179, 224, and 254;

Positions 89, 177, 179, 242, and 254;

Positions 89, 177, 180, 181, and 220;

Positions 89, 177, 180, 181, and 224;

Positions 89, 177, 180, 181, and 242;

Positions 89, 177, 180, 181, and 254;

Positions 89, 177, 180, 220, and 224;

Positions 89, 177, 180, 220, and 242;

Positions 89, 177, 180, 220, and 254;

Positions 89, 177, 180, 224, and 242;

Positions 89, 177, 180, 224, and 254;

Positions 89, 177, 180, 242, and 254;

Positions 89, 177, 181, 220, and 224;

Positions 89, 177, 181, 220, and 242;

Positions 89, 177, 181, 220, and 254;

Positions 89, 177, 181, 224, and 242;

Positions 89, 177, 181, 224, and 254;

Positions 89, 177, 181, 242, and 254;

Positions 89, 177, 220, 224, and 242;

Positions 89, 177, 220, 224, and 254;

Positions 89, 177, 220, 242, and 254;

Positions 89, 179, 181, 220, and 224;

Positions 89, 179, 181, 220, and 242;

Positions 89, 179, 181, 220, and 254;

Positions 89, 179, 181, 224, and 242;

Positions 89, 179, 181, 242, and 254;

Positions 89, 179, 220, 224, and 242;

Positions 89, 179, 220, 224, and 254;

Positions 89, 179, 220, 242, and 254;

Positions 89, 180, 181, 220, and 224;

Positions 89, 180, 181, 220, and 242;

Positions 89, 180, 181, 220, and 254;

Positions 89, 180, 181, 224, and 242;

Positions 89, 180, 181, 242, and 254;

Positions 89, 180, 220, 224, and 242;

Positions 89, 180, 220, 224, and 254;

Positions 89, 180, 220, 242, and 254;

Positions 89, 181, 220, 224, and 242;

Positions 89, 181, 220, 224, and 254;

Positions 89, 181, 220, 242, and 254;

Positions 89, 220, 224, 242, and 254;

Positions 129, 177, 179, 181, and 220;

Positions 129, 177, 179, 181, and 224;

Positions 129, 177, 179, 181, and 242;

Positions 129, 177, 179, 181, and 254;

Positions 129, 177, 179, 220, and 224;

Positions 129, 177, 179, 220, and 242;

Positions 129, 177, 179, 220, and 254;

Positions 129, 177, 179, 224, and 242;

Positions 129, 177, 179, 224, and 254;

Positions 129, 177, 179, 242, and 254;

Positions 129, 177, 180, 181, and 220;

Positions 129, 177, 180, 181, and 224;

Positions 129, 177, 180, 181, and 242;

Positions 129, 177, 180, 181, and 254;

Positions 129, 177, 180, 220, and 224;

Positions 129, 177, 180, 220, and 242;

Positions 129, 177, 180, 220, and 254;

Positions 129, 177, 180, 224, and 242;

Positions 129, 177, 180, 224, and 254;

Positions 129, 177, 180, 242, and 254;

Positions 129, 177, 181, 220, and 224;

Positions 129, 177, 181, 220, and 242;

Positions 129, 177, 181, 220, and 254;

Positions 129, 177, 181, 224, and 242;

Positions 129, 177, 181, 224, and 254;

Positions 129, 177, 181, 242, and 254;

Positions 129, 179, 181, 220, and 224;

Positions 129, 179, 181, 220, and 242;

Positions 129, 179, 181, 220, and 254;

Positions 129, 179, 181, 224, and 242;

Positions 129, 179, 181, 224, and 254;

Positions 129, 179, 181, 242, and 254;

Positions 129, 179, 220, 224, and 242;

Positions 129, 179, 220, 224, and 254;

Positions 129, 179, 220, 242, and 254;

Positions 129, 179, 224, 242, and 254;

Positions 129, 180, 181, 220, and 224;

Positions 129, 180, 181, 220, and 242;

Positions 129, 180, 181, 220, and 254;

Positions 129, 180, 181, 224, and 242;

Positions 129, 180, 181, 224, and 254;

Positions 129, 180, 181, 242, and 254;

Positions 129, 180, 220, 224, and 242;

Positions 129, 180, 220, 224, and 254;

Positions 129, 180, 220, 242, and 254;

Positions 129, 180, 224, 242, and 254;

Positions 129, 181, 220, 224, and 242;

Positions 129, 181, 220, 224, and 254;

Positions 129, 181, 220, 242, and 254;

Positions 129, 181, 224, 242, and 254;

Positions 129, 220, 224, 242, and 254;

Positions 177, 179, 181, 220, and 224;

Positions 177, 179, 181, 220, and 242;

Positions 177, 179, 181, 220, and 254;

Positions 177, 179, 181, 224, and 242;

Positions 177, 179, 181, 224, and 254;

Positions 177, 179, 181, 242, and 254;

Positions 177, 179, 220, 224, and 242;

Positions 177, 179, 220, 224, and 254;

Positions 177, 179, 220, 242, and 254;

Positions 177, 179, 224, 242, and 254;

Positions 177, 180, 181, 220, and 224;

Positions 177, 180, 181, 220, and 242;

Positions 177, 180, 181, 220, and 254;

Positions 177, 180, 181, 224, and 242;

Positions 177, 180, 181, 224, and 254;

Positions 177, 180, 181, 242, and 254;

Positions 177, 180, 220, 224, and 242;

Positions 177, 180, 220, 224, and 254;

Positions 177, 180, 224, 242, and 254;

Positions 177, 181, 220, 224, and 242;

Positions 177, 181, 220, 224, and 254;

Positions 177, 181, 224, 242, and 254;

Positions 177, 220, 224, 242, and 254;

Positions 179, 181, 220, 224, and 242;

Positions 179, 181, 220, 224, and 254;

Positions 179, 181, 220, 242, and 254;

Positions 179, 181, 224, 242, and 254;

Positions 179, 220, 224, 242, and 254;

Positions 180, 181, 220, 224, and 242;

Positions 180, 181, 220, 224, and 254;

Positions 180, 181, 220, 242, and 254;

Positions 180, 181, 224, 242, and 254;

Positions 180, 220, 224, 242, and 254; and

Positions 181, 220, 224, 242 and 254.

Paragraph 38. The variant of any of paragraphs 1-31, comprising asubstitution at six positions corresponding to any of positions 59, 89,91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184,208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and 427, inparticular, at the positions corresponding to positions 129, 177, 179,208, 242, and 284 or positions 129, 177, 179, 220, 224, and 254.Paragraph 39. The variant of paragraph 38, wherein the six positions areselected from the group consisting of:

Positions 59, 89, 129, 177, 179, and 181;

Positions 59, 89, 129, 177, 179, and 220;

Positions 59, 89, 129, 177, 179, and 224;

Positions 59, 89, 129, 177, 179, and 242;

Positions 59, 89, 129, 177, 179, and 254;

Positions 59, 89, 129, 177, 180, and 181;

Positions 59, 89, 129, 177, 180, and 220;

Positions 59, 89, 129, 177, 180, and 224;

Positions 59, 89, 129, 177, 180, and 242;

Positions 59, 89, 129, 177, 180, and 254;

Positions 59, 89, 129, 177, 181, and 220;

Positions 59, 89, 129, 177, 181, and 224;

Positions 59, 89, 129, 177, 181, and 242;

Positions 59, 89, 129, 177, 181, and 254;

Positions 59, 89, 129, 177, 220, and 224;

Positions 59, 89, 129, 177, 220, and 242;

Positions 59, 89, 129, 177, 220, and 254;

Positions 59, 89, 129, 177, 224, and 242;

Positions 59, 89, 129, 177, 224, and 254;

Positions 59, 89, 129, 177, 242, and 254;

Positions 59, 89, 129, 179, 181, and 220;

Positions 59, 89, 129, 179, 181, and 224;

Positions 59, 89, 129, 179, 181, and 242;

Positions 59, 89, 129, 179, 181, and 254;

Positions 59, 89, 129, 179, 220, and 224;

Positions 59, 89, 129, 179, 220, and 242;

Positions 59, 89, 129, 179, 220, and 254;

Positions 59, 89, 129, 179, 224, and 242;

Positions 59, 89, 129, 179, 224, and 254;

Positions 59, 89, 129, 179, 242, and 254;

Positions 59, 89, 129, 180, 181, and 220;

Positions 59, 89, 129, 180, 181, and 224;

Positions 59, 89, 129, 180, 181, and 242;

Positions 59, 89, 129, 180, 181, and 254;

Positions 59, 89, 129, 180, 220, and 224;

Positions 59, 89, 129, 180, 220, and 242;

Positions 59, 89, 129, 180, 220, and 254;

Positions 59, 89, 129, 180, 224, and 242;

Positions 59, 89, 129, 180, 224, and 254;

Positions 59, 89, 129, 180, 242, and 254;

Positions 59, 89, 129, 181, 220, and 224;

Positions 59, 89, 129, 181, 220, and 242;

Positions 59, 89, 129, 181, 220, and 254;

Positions 59, 89, 129, 181, 224, and 242;

Positions 59, 89, 129, 181, 224, and 254;

Positions 59, 89, 129, 181, 242, and 254;

Positions 59, 89, 129, 220, 224, and 242;

Positions 59, 89, 129, 220, 224, and 254;

Positions 59, 89, 129, 220, 242, and 254;

Positions 59, 89, 129, 224, 242, and 254;

Positions 59, 89, 177, 179, 181, and 220;

Positions 59, 89, 177, 179, 181, and 224;

Positions 59, 89, 177, 179, 181, and 242;

Positions 59, 89, 177, 179, 181, and 254;

Positions 59, 89, 177, 179, 220, and 224;

Positions 59, 89, 177, 179, 220, and 242;

Positions 59, 89, 177, 179, 220, and 254;

Positions 59, 89, 177, 179, 224, and 242;

Positions 59, 89, 177, 179, 224, and 254;

Positions 59, 89, 177, 179, 242, and 254;

Positions 59, 89, 177, 180, 181, and 220;

Positions 59, 89, 177, 180, 181, and 224;

Positions 59, 89, 177, 180, 181, and 242;

Positions 59, 89, 177, 180, 181, and 254;

Positions 59, 89, 177, 180, 220, and 224;

Positions 59, 89, 177, 180, 220, and 242;

Positions 59, 89, 177, 180, 220, and 254;

Positions 59, 89, 177, 180, 224, and 242;

Positions 59, 89, 177, 180, 224, and 254;

Positions 59, 89, 177, 180, 242, and 254;

Positions 59, 89, 177, 181, 220, and 224;

Positions 59, 89, 177, 181, 220, and 242;

Positions 59, 89, 177, 181, 220, and 254;

Positions 59, 89, 177, 181, 224, and 242;

Positions 59, 89, 177, 181, 224, and 254;

Positions 59, 89, 177, 181, 242, and 254;

Positions 59, 89, 177, 220, 224, and 242;

Positions 59, 89, 177, 220, 224, and 254;

Positions 59, 89, 177, 220, 242, and 254;

Positions 59, 89, 179, 181, 220, and 224;

Positions 59, 89, 179, 181, 220, and 242;

Positions 59, 89, 179, 181, 220, and 254;

Positions 59, 89, 179, 181, 224, and 242;

Positions 59, 89, 179, 181, 224, and 254;

Positions 59, 89, 179, 181, 242, and 254;

Positions 59, 89, 179, 220, 224, and 242;

Positions 59, 89, 179, 220, 224, and 254;

Positions 59, 89, 179, 220, 242, and 254;

Positions 59, 89, 180, 181, 220, and 224;

Positions 59, 89, 180, 181, 220, and 242;

Positions 59, 89, 180, 181, 220, and 254;

Positions 59, 89, 180, 181, 224, and 242;

Positions 59, 89, 180, 181, 224, and 254;

Positions 59, 89, 180, 181, 242, and 254;

Positions 59, 89, 180, 220, 224, and 242;

Positions 59, 89, 180, 220, 224, and 254;

Positions 59, 89, 180, 220, 242, and 254;

Positions 59, 89, 181, 220, 224, and 242;

Positions 59, 89, 181, 220, 224, and 254;

Positions 59, 89, 181, 220, 242, and 254;

Positions 59, 89, 220, 224, 242, and 254;

Positions 59, 129, 177, 179, 181, and 220;

Positions 59, 129, 177, 179, 181, and 224;

Positions 59, 129, 177, 179, 181, and 242;

Positions 59, 129, 177, 179, 181, and 254;

Positions 59, 129, 177, 179, 208, and 284;

Positions 59, 129, 177, 179, 220, and 224;

Positions 59, 129, 177, 179, 220, and 242;

Positions 59, 129, 177, 179, 220, and 254;

Positions 59, 129, 177, 179, 224, and 242;

Positions 59, 129, 177, 179, 224, and 254;

Positions 59, 129, 177, 179, 242, and 254;

Positions 59, 129, 177, 180, 181, and 220;

Positions 59, 129, 177, 180, 181, and 224;

Positions 59, 129, 177, 180, 181, and 242;

Positions 59, 129, 177, 180, 181, and 254;

Positions 59, 129, 177, 180, 220, and 224;

Positions 59, 129, 177, 180, 220, and 242;

Positions 59, 129, 177, 180, 220, and 254;

Positions 59, 129, 177, 180, 224, and 242;

Positions 59, 129, 177, 180, 224, and 254;

Positions 59, 129, 177, 180, 242, and 254;

Positions 59, 129, 177, 181, 220, and 224;

Positions 59, 129, 177, 181, 220, and 242;

Positions 59, 129, 177, 181, 220, and 254;

Positions 59, 129, 177, 181, 224, and 242;

Positions 59, 129, 177, 181, 224, and 254;

Positions 59, 129, 177, 181, 242, and 254;

Positions 59, 129, 177, 220, 224, and 242;

Positions 59, 129, 177, 220, 224, and 254;

Positions 59, 129, 177, 224, 242, and 254;

Positions 59, 129, 179, 181, 220, and 224;

Positions 59, 129, 179, 181, 220, and 242;

Positions 59, 129, 179, 181, 220, and 254;

Positions 59, 129, 179, 220, 224, and 242;

Positions 59, 129, 179, 220, 224, and 254;

Positions 59, 129, 179, 220, 242, and 254;

Positions 59, 129, 179, 224, 242, and 254;

Positions 59, 129, 180, 181, 220, and 224;

Positions 59, 129, 180, 181, 220, and 242;

Positions 59, 129, 180, 181, 220, and 254;

Positions 59, 129, 180, 220, 224, and 242;

Positions 59, 129, 180, 220, 224, and 254;

Positions 59, 129, 180, 220, 242, and 254;

Positions 59, 129, 180, 224, 242, and 254;

Positions 59, 129, 181, 220, 224, and 242;

Positions 59, 129, 181, 220, 224, and 254;

Positions 59, 129, 181, 220, 242, and 254;

Positions 59, 129, 181, 224, 242, and 254;

Positions 59, 129, 220, 224, 242, and 254;

Positions 59, 177, 179, 181, 220, and 224;

Positions 59, 177, 179, 181, 220, and 242;

Positions 59, 177, 179, 181, 220, and 254;

Positions 59, 177, 179, 181, 224, and 242;

Positions 59, 177, 179, 181, 224, and 254;

Positions 59, 177, 179, 181, 242, and 254;

Positions 59, 177, 179, 220, 224, and 242;

Positions 59, 177, 179, 220, 224, and 254;

Positions 59, 177, 179, 220, 242, and 254;

Positions 59, 177, 179, 224, 242, and 254;

Positions 59, 177, 180, 181, 220, and 224;

Positions 59, 177, 180, 181, 220, and 242;

Positions 59, 177, 180, 181, 220, and 254;

Positions 59, 177, 180, 181, 224, and 242;

Positions 59, 177, 180, 181, 224, and 254;

Positions 59, 177, 180, 181, 242, and 254;

Positions 59, 177, 180, 220, 224, and 242;

Positions 59, 177, 180, 220, 224, and 254;

Positions 59, 177, 180, 220, 242, and 254;

Positions 59, 177, 180, 224, 242, and 254;

Positions 59, 177, 181, 220, 224, and 242;

Positions 59, 177, 181, 220, 224, and 254;

Positions 59, 177, 181, 220, 242, and 254;

Positions 59, 177, 181, 224, 242, and 254;

Positions 59, 177, 220, 224, 242, and 254;

Positions 59, 179, 181, 220, 224, and 242;

Positions 59, 179, 181, 220, 224, and 254;

Positions 59, 179, 181, 220, 242, and 254;

Positions 59, 179, 181, 224, 242, and 254;

Positions 59, 179, 220, 224, 242, and 254;

Positions 59, 180, 181, 220, 224, and 242;

Positions 59, 180, 181, 220, 224, and 254;

Positions 59, 180, 181, 220, 242, and 254;

Positions 59, 180, 181, 224, 242, and 254;

Positions 59, 180, 220, 224, 242, and 254;

Positions 59, 181, 220, 224, 242, and 254;

Positions 59, 208, 220, 224, 254, and 284;

Positions 89, 129, 177, 179, 181, and 220;

Positions 89, 129, 177, 179, 181, and 224;

Positions 89, 129, 177, 179, 181, and 242;

Positions 89, 129, 177, 179, 181, and 254;

Positions 89, 129, 177, 179, 220, and 224;

Positions 89, 129, 177, 179, 220, and 242;

Positions 89, 129, 177, 179, 220, and 254;

Positions 89, 129, 177, 179, 224, and 242;

Positions 89, 129, 177, 179, 224, and 254;

Positions 89, 129, 177, 179, 242, and 254;

Positions 89, 129, 177, 180, 181, and 220;

Positions 89, 129, 177, 180, 181, and 224;

Positions 89, 129, 177, 180, 181, and 242;

Positions 89, 129, 177, 180, 181, and 254;

Positions 89, 129, 177, 180, 220, and 224;

Positions 89, 129, 177, 180, 220, and 242;

Positions 89, 129, 177, 180, 220, and 254;

Positions 89, 129, 177, 180, 224, and 242;

Positions 89, 129, 177, 180, 224, and 254;

Positions 89, 129, 177, 180, 242, and 254;

Positions 89, 129, 177, 181, 220, and 224;

Positions 89, 129, 177, 181, 220, and 242;

Positions 89, 129, 177, 181, 220, and 254;

Positions 89, 129, 177, 181, 224, and 242;

Positions 89, 129, 177, 181, 224, and 254;

Positions 89, 129, 177, 181, 242, and 254;

Positions 89, 129, 177, 220, 224, and 242;

Positions 89, 129, 177, 220, 224, and 254;

Positions 89, 129, 177, 220, 242, and 254;

Positions 89, 129, 177, 224, 242, and 254;

Positions 89, 129, 179, 181, 220, and 224;

Positions 89, 129, 179, 181, 220, and 242;

Positions 89, 129, 179, 181, 220, and 254;

Positions 89, 129, 179, 181, 224, and 242;

Positions 89, 129, 179, 181, 224, and 254;

Positions 89, 129, 179, 181, 242, and 254;

Positions 89, 129, 179, 220, 224, and 242;

Positions 89, 129, 179, 220, 224, and 254;

Positions 89, 129, 179, 220, 242, and 254;

Positions 89, 129, 179, 224, 242, and 254;

Positions 89, 129, 180, 220, 224, and 242;

Positions 89, 129, 180, 220, 224, and 254;

Positions 89, 129, 180, 220, 242, and 254;

Positions 89, 129, 180, 224, 242, and 254;

Positions 89, 129, 181, 220, 224, and 242;

Positions 89, 129, 181, 220, 224, and 254;

Positions 89, 129, 181, 220, 242, and 254;

Positions 89, 129, 181, 224, 242, and 254;

Positions 89, 129, 220, 224, 242, and 254;

Positions 89, 177, 179, 181, 220, and 224;

Positions 89, 177, 179, 181, 220, and 242;

Positions 89, 177, 179, 181, 220, and 254;

Positions 89, 177, 179, 181, 224, and 242;

Positions 89, 177, 179, 181, 224, and 254;

Positions 89, 177, 179, 181, 242, and 254;

Positions 89, 177, 179, 220, 224, and 242;

Positions 89, 177, 179, 220, 224, and 254;

Positions 89, 177, 179, 220, 242, and 254;

Positions 89, 177, 179, 224, 242, and 254;

Positions 89, 177, 180, 181, 220, and 224;

Positions 89, 177, 180, 181, 220, and 242;

Positions 89, 177, 180, 181, 220, and 254;

Positions 89, 177, 180, 181, 224, and 242;

Positions 89, 177, 180, 181, 224, and 254;

Positions 89, 177, 180, 181, 242, and 254;

Positions 89, 177, 180, 220, 224, and 242;

Positions 89, 177, 180, 220, 224, and 254;

Positions 89, 177, 180, 220, 242, and 254;

Positions 89, 177, 180, 224, 242, and 254;

Positions 89, 177, 181, 220, 224, and 242;

Positions 89, 177, 181, 220, 224, and 254;

Positions 89, 177, 181, 220, 242, and 254;

Positions 89, 177, 181, 224, 242, and 254;

Positions 89, 177, 220, 224, 242, and 254;

Positions 89, 179, 181, 220, 224, and 242;

Positions 89, 179, 181, 220, 224, and 254;

Positions 89, 179, 181, 220, 242, and 254;

Positions 89, 179, 181, 224, 242, and 254;

Positions 89, 179, 220, 224, 242, and 254;

Positions 89, 180, 181, 220, 224, and 242;

Positions 89, 180, 181, 220, 224, and 254;

Positions 89, 180, 181, 220, 242, and 254;

Positions 89, 180, 181, 224, 242, and 254;

Positions 89, 180, 220, 224, 242, and 254;

Positions 89, 181, 220, 224, 242, and 254;

Positions 129, 177, 179, 181, 220, and 224;

Positions 129, 177, 179, 181, 220, and 242;

Positions 129, 177, 179, 181, 220, and 254;

Positions 129, 177, 179, 181, 224, and 242;

Positions 129, 177, 179, 181, 224, and 254;

Positions 129, 177, 179, 181, 242, and 254;

Positions 129, 177, 179, 220, 224, and 242;

Positions 129, 177, 179, 220, 224, and 254;

Positions 129, 177, 179, 220, 242, and 254;

Positions 129, 177, 179, 224, 242, and 254;

Positions 129, 177, 180, 181, 220, and 224;

Positions 129, 177, 180, 181, 220, and 242;

Positions 129, 177, 180, 181, 220, and 254;

Positions 129, 177, 180, 181, 224, and 242;

Positions 129, 177, 180, 181, 224, and 254;

Positions 129, 177, 180, 181, 242, and 254;

Positions 129, 177, 180, 220, 224, and 242;

Positions 129, 177, 180, 220, 224, and 254;

Positions 129, 177, 180, 220, 242, and 254;

Positions 129, 177, 180, 224, 242, and 254;

Positions 129, 177, 181, 220, 224, and 242;

Positions 129, 177, 181, 220, 224, and 254;

Positions 129, 177, 181, 220, 242, and 254;

Positions 129, 177, 181, 224, 242, and 254;

Positions 129, 177, 200, 224, 242, and 254;

Positions 129, 179, 181, 220, 224, and 242;

Positions 129, 179, 181, 220, 224, and 254;

Positions 129, 179, 181, 220, 242, and 254;

Positions 129, 179, 181, 224, 242, and 254;

Positions 129, 179, 220, 224, 242, and 254;

Positions 129, 180, 181, 220, 224, and 242;

Positions 129, 180, 181, 220, 224, and 254;

Positions 129, 180, 181, 220, 242, and 254;

Positions 129, 180, 181, 224, 242, and 254;

Positions 129, 180, 220, 224, 242, and 254;

Positions 129, 181, 220, 224, 242, and 254;

Positions 177, 179, 181, 220, 224, and 242;

Positions 177, 179, 181, 220, 224, and 254;

Positions 177, 179, 181, 224, 242, and 254;

Positions 177, 179, 220, 224, 242, and 254;

Positions 177, 180, 181, 220, 224, and 242;

Positions 177, 180, 181, 220, 224, and 254;

Positions 177, 180, 181, 224, 242, and 254;

Positions 177, 180, 220, 224, 242, and 254;

Positions 177, 181, 220, 224, 242, and 254;

Positions 179, 181, 220, 224, 242, and 254; and

Positions 180, 181, 220, 224, 242, and 254.

Paragraph 40. The variant of any of paragraphs 1-31, comprising asubstitution at seven positions corresponding to any of positions 59,89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181,184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and427.Paragraph 41. The variant of paragraph 40, wherein the seven positionsare selected from the group consisting of:

Positions 59, 89, 129, 177, 179, 181, and 220;

Positions 59, 89, 129, 177, 179, 181, and 224;

Positions 59, 89, 129, 177, 179, 181, and 242;

Positions 59, 89, 129, 177, 179, 181, and 254;

Positions 59, 89, 129, 177, 179, 220, and 224;

Positions 59, 89, 129, 177, 179, 220, and 242;

Positions 59, 89, 129, 177, 179, 220, and 254;

Positions 59, 89, 129, 177, 179, 224, and 242;

Positions 59, 89, 129, 177, 179, 224, and 254;

Positions 59, 89, 129, 177, 179, 242, and 254;

Positions 59, 89, 129, 177, 180, 181, and 220;

Positions 59, 89, 129, 177, 180, 181, and 224;

Positions 59, 89, 129, 177, 180, 181, and 242;

Positions 59, 89, 129, 177, 180, 181, and 254;

Positions 59, 89, 129, 177, 180, 220, and 224;

Positions 59, 89, 129, 177, 180, 220, and 242;

Positions 59, 89, 129, 177, 180, 220, and 254;

Positions 59, 89, 129, 177, 180, 224, and 242;

Positions 59, 89, 129, 177, 180, 224, and 254;

Positions 59, 89, 129, 177, 180, 242, and 254;

Positions 59, 89, 129, 177, 181, 220, and 224;

Positions 59, 89, 129, 177, 181, 220, and 242;

Positions 59, 89, 129, 177, 181, 220, and 254;

Positions 59, 89, 129, 177, 181, 224, and 242;

Positions 59, 89, 129, 177, 181, 224, and 254;

Positions 59, 89, 129, 177, 181, 242, and 254;

Positions 59, 89, 129, 177, 220, 224, and 242;

Positions 59, 89, 129, 177, 220, 224, and 254;

Positions 59, 89, 129, 177, 224, 242, and 254;

Positions 59, 89, 129, 179, 181, 220, and 224;

Positions 59, 89, 129, 179, 181, 220, and 242;

Positions 59, 89, 129, 179, 181, 220, and 254;

Positions 59, 89, 129, 179, 181, 224, and 242;

Positions 59, 89, 129, 179, 181, 224, and 254;

Positions 59, 89, 129, 179, 181, 242, and 254;

Positions 59, 89, 129, 179, 220, 224, and 242;

Positions 59, 89, 129, 179, 220, 224, and 254;

Positions 59, 89, 129, 179, 220, 242, and 254;

Positions 59, 89, 129, 179, 224, 242, and 254;

Positions 59, 89, 129, 180, 181, 220, and 224;

Positions 59, 89, 129, 180, 181, 220, and 242;

Positions 59, 89, 129, 180, 181, 220, and 254;

Positions 59, 89, 129, 180, 181, 224, and 242;

Positions 59, 89, 129, 180, 181, 224, and 254;

Positions 59, 89, 129, 180, 181, 242, and 254;

Positions 59, 89, 129, 180, 220, 224, and 242;

Positions 59, 89, 129, 180, 220, 224, and 254;

Positions 59, 89, 129, 180, 220, 242, and 254;

Positions 59, 89, 129, 180, 224, 242, and 254;

Positions 59, 89, 129, 181, 220, 224, and 242;

Positions 59, 89, 129, 181, 220, 224, and 254;

Positions 59, 89, 129, 181, 220, 242, and 254;

Positions 59, 89, 129, 181, 224, 242, and 254;

Positions 59, 89, 129, 220, 224, 242, and 254;

Positions 59, 89, 177, 179, 181, 220, and 224;

Positions 59, 89, 177, 179, 181, 220, and 242;

Positions 59, 89, 177, 179, 181, 220, and 254;

Positions 59, 89, 177, 179, 181, 224, and 242;

Positions 59, 89, 177, 179, 181, 224, and 254;

Positions 59, 89, 177, 179, 181, 242, and 254;

Positions 59, 89, 177, 179, 220, 224, and 242;

Positions 59, 89, 177, 179, 220, 224, and 254;

Positions 59, 89, 177, 179, 224, 242, and 254;

Positions 59, 89, 177, 180, 181, 220, and 224;

Positions 59, 89, 177, 180, 181, 220, and 242;

Positions 59, 89, 177, 180, 181, 220, and 254;

Positions 59, 89, 177, 180, 181, 224, and 242;

Positions 59, 89, 177, 180, 181, 224, and 254;

Positions 59, 89, 177, 180, 181, 242, and 254;

Positions 59, 89, 177, 180, 220, 224, and 242;

Positions 59, 89, 177, 180, 220, 224, and 254;

Positions 59, 89, 177, 180, 224, 242, and 254;

Positions 59, 89, 177, 181, 220, 224, and 242;

Positions 59, 89, 177, 181, 220, 224, and 254;

Positions 59, 89, 177, 181, 220, 242, and 254;

Positions 59, 89, 177, 181, 224, 242, and 254;

Positions 59, 89, 177, 220, 224, 242, and 254;

Positions 59, 89, 179, 181, 220, 224, and 242;

Positions 59, 89, 179, 181, 220, 224, and 254;

Positions 59, 89, 179, 181, 220, 242, and 254;

Positions 59, 89, 179, 220, 224, 242, and 254;

Positions 59, 89, 180, 181, 220, 224, and 242;

Positions 59, 89, 180, 181, 220, 224, and 254;

Positions 59, 89, 180, 181, 220, 242, and 254;

Positions 59, 89, 180, 220, 224, 242, and 254;

Positions 59, 89, 181, 220, 224, 242, and 254;

Positions 59, 129, 177, 179, 181, 220, and 224;

Positions 59, 129, 177, 179, 181, 220, and 242;

Positions 59, 129, 177, 179, 181, 220, and 254;

Positions 59, 129, 177, 179, 181, 224, and 242;

Positions 59, 129, 177, 179, 181, 224, and 254;

Positions 59, 129, 177, 179, 181, 242, and 254;

Positions 59, 129, 177, 179, 220, 224, and 242;

Positions 59, 129, 177, 179, 220, 224, and 254;

Positions 59, 129, 177, 179, 220, 242, and 254;

Positions 59, 129, 177, 179, 224, 242, and 254;

Positions 59, 129, 177, 180, 181, 220, and 224;

Positions 59, 129, 177, 180, 181, 220, and 242;

Positions 59, 129, 177, 180, 181, 220, and 254;

Positions 59, 129, 177, 180, 181, 224, and 242;

Positions 59, 129, 177, 180, 181, 224, and 254;

Positions 59, 129, 177, 180, 181, 242, and 254;

Positions 59, 129, 177, 180, 220, 224, and 242;

Positions 59, 129, 177, 180, 220, 224, and 254;

Positions 59, 129, 177, 180, 220, 242, and 254;

Positions 59, 129, 177, 180, 224, 242, and 254;

Positions 59, 129, 177, 181, 220, 224, and 242;

Positions 59, 129, 177, 181, 220, 224, and 254;

Positions 59, 129, 177, 181, 220, 242, and 254;

Positions 59, 129, 177, 181, 224, 242, and 254;

Positions 59, 129, 177, 220, 224, 242, and 254;

Positions 59, 177, 179, 181, 220, 224, and 242;

Positions 59, 177, 179, 181, 220, 224, and 254;

Positions 59, 177, 179, 181, 220, 242, and 254;

Positions 59, 177, 179, 181, 224, 242, and 254;

Positions 59, 177, 179, 220, 224, 242, and 254;

Positions 59, 177, 180, 181, 220, 224, and 242;

Positions 59, 177, 180, 181, 220, 224, and 254;

Positions 59, 177, 180, 181, 220, 242, and 254;

Positions 59, 177, 180, 181, 224, 242, and 254;

Positions 59, 177, 180, 220, 224, 242, and 254;

Positions 59, 177, 181, 220, 224, 242, and 254;

Positions 59, 179, 181, 220, 224, 242, and 254;

Positions 59, 180, 181, 220, 224, 242, and 254;

Positions 89, 129, 177, 179, 181, 220, and 224;

Positions 89, 129, 177, 179, 181, 220, and 242;

Positions 89, 129, 177, 179, 181, 220, and 254;

Positions 89, 129, 177, 179, 181, 224, and 242;

Positions 89, 129, 177, 179, 181, 224, and 254;

Positions 89, 129, 177, 179, 181, 242, and 254;

Positions 89, 129, 177, 179, 220, 224, and 242;

Positions 89, 129, 177, 179, 220, 224, and 254;

Positions 89, 129, 177, 179, 220, 242, and 254;

Positions 89, 129, 177, 179, 224, 242, and 254;

Positions 89, 129, 177, 180, 181, 220, and 224;

Positions 89, 129, 177, 180, 181, 220, and 242;

Positions 89, 129, 177, 180, 181, 220, and 254;

Positions 89, 129, 177, 180, 181, 224, and 242;

Positions 89, 129, 177, 180, 181, 224, and 254;

Positions 89, 129, 177, 180, 181, 242, and 254;

Positions 89, 129, 177, 180, 220, 224, and 242;

Positions 89, 129, 177, 180, 220, 224, and 254;

Positions 89, 129, 177, 180, 220, 242, and 254;

Positions 89, 129, 177, 180, 224, 242, and 254;

Positions 89, 129, 177, 181, 220, 224, and 242;

Positions 89, 129, 177, 181, 220, 224, and 254;

Positions 89, 129, 177, 181, 220, 242, and 254;

Positions 89, 129, 177, 181, 224, 242, and 254;

Positions 89, 129, 177, 220, 224, 242, and 254;

Positions 89, 129, 179, 181, 220, 224, and 242;

Positions 89, 129, 179, 181, 220, 224, and 254;

Positions 89, 129, 179, 181, 220, 242, and 254;

Positions 89, 129, 179, 181, 224, 242, and 254;

Positions 89, 129, 179, 220, 224, 242, and 254;

Positions 89, 129, 180, 181, 220, 224, and 242;

Positions 89, 129, 180, 181, 220, 224, and 254;

Positions 89, 129, 180, 181, 220, 242, and 254;

Positions 89, 129, 180, 181, 224, 242, and 254;

Positions 89, 129, 180, 220, 224, 242, and 254;

Positions 89, 129, 181, 220, 224, 242, and 254;

Positions 89, 177, 179, 181, 220, 224, and 242;

Positions 89, 177, 179, 181, 220, 224, and 254;

Positions 89, 177, 179, 181, 220, 242, and 254;

Positions 89, 177, 179, 181, 224, 242, and 254;

Positions 89, 177, 179, 220, 224, 242, and 254;

Positions 89, 177, 180, 181, 220, 224, and 242;

Positions 89, 177, 180, 181, 220, 224, and 254;

Positions 89, 177, 180, 181, 220, 242, and 254;

Positions 89, 177, 180, 181, 224, 242, and 254;

Positions 89, 177, 180, 220, 224, 242, and 254;

Positions 89, 177, 181, 220, 224, 242, and 254;

Positions 89, 179, 181, 220, 224, 242, and 254;

Positions 89, 180, 181, 220, 224, 242, and 254;

Positions 129, 177, 179, 181, 220, 224, and 242;

Positions 129, 177, 179, 181, 220, 224, and 254;

Positions 129, 177, 179, 181, 220, 242, and 254;

Positions 129, 177, 179, 181, 224, 242, and 254;

Positions 129, 177, 179, 220, 224, 242, and 254;

Positions 129, 177, 180, 181, 220, 224, and 242;

Positions 129, 177, 180, 181, 220, 224, and 254;

Positions 129, 177, 180, 181, 220, 242, and 254;

Positions 129, 177, 180, 181, 224, 242, and 254;

Positions 129, 177, 180, 220, 224, 242, and 254;

Positions 129, 177, 181, 220, 224, 242, and 254;

Positions 129, 179, 181, 220, 224, 242, and 254;

Positions 129, 180, 181, 220, 224, 242, and 254;

Positions 177, 179, 181, 220, 224, 242, and 254; and

Positions 177, 180, 181, 220, 224, 242, and 254.

Paragraph 42. The variant of any of paragraphs 1-31, comprising asubstitution at eight positions corresponding to any of positions 59,89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181,184, 208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and427.Paragraph 43. The variant of paragraph 42, wherein the eight positionsare selected from the group consisting of:

Positions 59, 89, 129, 177, 179, 181, 220, and 224;

Positions 59, 89, 129, 177, 179, 181, 220, and 242;

Positions 59, 89, 129, 177, 179, 181, 220, and 254;

Positions 59, 89, 129, 177, 179, 181, 224, and 242;

Positions 59, 89, 129, 177, 179, 181, 224, and 254;

Positions 59, 89, 129, 177, 179, 181, 242, and 254;

Positions 59, 89, 129, 177, 179, 220, 224, and 242;

Positions 59, 89, 129, 177, 179, 220, 224, and 254;

Positions 59, 89, 129, 177, 179, 220, 242, and 254;

Positions 59, 89, 129, 177, 179, 224, 242, and 254;

Positions 59, 89, 129, 177, 180, 181, 220, and 224;

Positions 59, 89, 129, 177, 180, 181, 220, and 242;

Positions 59, 89, 129, 177, 180, 181, 220, and 254;

Positions 59, 89, 129, 177, 180, 181, 224, and 242;

Positions 59, 89, 129, 177, 180, 181, 224, and 254;

Positions 59, 89, 129, 177, 180, 181, 242, and 254;

Positions 59, 89, 129, 177, 180, 220, 224, and 242;

Positions 59, 89, 129, 177, 180, 220, 224, and 254;

Positions 59, 89, 129, 177, 180, 220, 242, and 254;

Positions 59, 89, 129, 177, 180, 224, 242, and 254;

Positions 59, 89, 129, 177, 181, 220, 224, and 242;

Positions 59, 89, 129, 177, 181, 220, 224, and 254;

Positions 59, 89, 129, 177, 181, 220, 242, and 254;

Positions 59, 89, 129, 177, 181, 224, 242, and 254;

Positions 59, 89, 129, 177, 220, 224, 242, and 254;

Positions 59, 89, 129, 179, 181, 220, 224, and 242;

Positions 59, 89, 129, 179, 181, 220, 224, and 254;

Positions 59, 89, 129, 179, 181, 220, 242, and 254;

Positions 59, 89, 129, 179, 181, 224, 242, and 254;

Positions 59, 89, 129, 179, 220, 224, 242, and 254;

Positions 59, 89, 129, 180, 181, 220, 224, and 242;

Positions 59, 89, 129, 180, 181, 220, 224, and 254;

Positions 59, 89, 129, 180, 181, 220, 242, and 254;

Positions 59, 89, 129, 180, 181, 224, 242, and 254;

Positions 59, 89, 129, 180, 220, 224, 242, and 254;

Positions 59, 89, 129, 181, 220, 224, 242, and 254;

Positions 59, 89, 177, 179, 181, 220, 224, and 242;

Positions 59, 89, 177, 179, 181, 220, 224, and 254;

Positions 59, 89, 177, 179, 181, 220, 242, and 254;

Positions 59, 89, 177, 179, 181, 224, 242, and 254;

Positions 59, 89, 177, 179, 220, 224, 242, and 254;

Positions 59, 89, 177, 180, 181, 220, 224, and 242;

Positions 59, 89, 177, 180, 181, 220, 224, and 254;

Positions 59, 89, 177, 180, 181, 220, 242, and 254;

Positions 59, 89, 177, 180, 181, 224, 242, and 254;

Positions 59, 89, 177, 180, 220, 224, 242, and 254;

Positions 59, 89, 177, 181, 220, 224, 242, and 254;

Positions 59, 89, 179, 181, 220, 224, 242, and 254;

Positions 59, 89, 180, 181, 220, 224, 242, and 254;

Positions 59, 129, 177, 179, 181, 220, 224, and 242;

Positions 59, 129, 177, 179, 181, 220, 224, and 254;

Positions 59, 129, 177, 179, 181, 224, 242, and 254;

Positions 59, 129, 177, 179, 220, 224, 242, and 254;

Positions 59, 129, 177, 180, 181, 220, 224, and 242;

Positions 59, 129, 177, 180, 181, 220, 224, and 254;

Positions 59, 129, 177, 180, 181, 224, 242, and 254;

Positions 59, 129, 177, 180, 220, 224, 242, and 254;

Positions 59, 129, 177, 181, 220, 224, 242, and 254;

Positions 59, 129, 179, 181, 220, 224, 242, and 254;

Positions 59, 129, 180, 181, 220, 224, 242, and 254;

Positions 59, 177, 179, 181, 220, 224, 242, and 254;

Positions 59, 177, 180, 181, 220, 224, 242, and 254;

Positions 89, 129, 177, 179, 181, 220, 224, and 242;

Positions 89, 129, 177, 179, 181, 220, 224, and 254;

Positions 89, 129, 177, 179, 181, 224, 242, and 254;

Positions 89, 129, 177, 179, 220, 224, 242, and 254;

Positions 89, 129, 177, 180, 181, 220, 224, and 242;

Positions 89, 129, 177, 180, 181, 220, 224, and 254;

Positions 89, 129, 177, 180, 181, 224, 242, and 254;

Positions 89, 129, 177, 180, 220, 224, 242, and 254;

Positions 89, 129, 177, 181, 220, 224, 242, and 254;

Positions 129, 177, 179, 181, 220, 224, 242, and 254; and

Positions 129, 177, 180, 181, 220, 224, 242, and 254.

Paragraph 44. The variant of any of paragraphs 1-31, comprising asubstitution at nine positions corresponding to any of positions 59, 89,91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184,208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and 427.Paragraph 45. The variant of paragraph 44, wherein the nine positionsare selected from the group consisting of:

Positions 59, 89, 129, 177, 179, 181, 220, 224, and 242;

Positions 59, 89, 129, 177, 179, 181, 220, 224, and 254;

Positions 59, 89, 129, 177, 179, 181, 220, 242, and 254;

Positions 59, 89, 129, 177, 179, 181, 224, 242, and 254;

Positions 59, 89, 129, 177, 179, 220, 224, 242, and 254;

Positions 59, 89, 129, 177, 180, 181, 220, 224, and 242;

Positions 59, 89, 129, 177, 180, 181, 220, 224, and 254;

Positions 59, 89, 129, 177, 180, 181, 220, 242, and 254;

Positions 59, 89, 129, 177, 180, 181, 224, 242, and 254;

Positions 59, 89, 129, 177, 180, 220, 224, 242, and 254;

Positions 59, 89, 129, 177, 181, 220, 224, 242, and 254;

Positions 59, 89, 129, 179, 181, 220, 224, 242, and 254;

Positions 59, 89, 129, 180, 181, 220, 224, 242, and 254;

Positions 59, 89, 177, 179, 181, 220, 224, 242, and 254;

Positions 59, 89, 177, 180, 181, 220, 224, 242, and 254;

Positions 59, 129, 177, 179, 181, 220, 224, 242, and 254;

Positions 59, 129, 177, 179, 208, 220, 224, 254, and 284;

Positions 89, 129, 177, 179, 181, 220, 224, 242, and 254; and

Positions 89, 129, 177, 180, 181, 220, 224, 242, and 254.

Paragraph 46. The variant of any of paragraphs 1-31, comprising asubstitution at ten positions corresponding to any of positions 59, 89,91, 96, 108, 112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184,208, 220, 224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and 427.Paragraph 47. The variant of paragraph 46, wherein the ten positions arethe positions corresponding to:

Positions 59, 89, 129, 177, 179, 181, 220, 224, 242, 254;

Positions 59, 89, 129, 177, 179, 208, 220, 224, 254, 284;

Positions 59, 89, 129, 177, 180, 181, 220, 224, 242, 254; and

Positions 59, 129, 177, 179, 208, 220, 224, 242, 254, 284.

Paragraph 48. The variant of any of paragraphs 1-47, which has 3-20,e.g., 3-10 and 6-10, alterations such as 3, 4, 5, 6, 7, 8, 9 or 10alterations.Paragraph 49. An isolated variant alpha-amylase, comprising orconsisting of a set of substitutions selected from the group consistingof:

V59A+G108A;

S242Q+M284V;

V59A+M284V;

G108A+M284V;

V59A+G108A+M284V;

V59A+G108A+S242Q+M284V;

E129V+K177L+R179E;

K220P+N224L+Q254S;

E129V+K177L+R179E+M284V;

V59A+E129V+K177L+R179E+H208Y+M284V;

V59A+H208Y+K220P+N224L+Q254S+M284V;

E129V+K177L+R179E+K220P+N224L+Q254S;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;

V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S+M284V;

V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;

V59A+Q89R+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S+M284V; and

V59A+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;

wherein the variant has at least 65% and less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6,and/or 7 and the variant has alpha-amylase activity.Paragraph 50. The variant of any of paragraphs 1-49, which has 3-20,e.g., 3-10 and 6-10, alterations such as 3, 4, 5, 6, 7, 8, 9 or 10alterations.Paragraph 51. The variant of paragraph 50, wherein the alterations aresubstitutions.Paragraph 52. The variant of any of paragraphs 1-50, which furthercomprises a deletion at one or more, e.g., two, three or four, positionscorresponding to positions 179, 180, 181 and 182.Paragraph 53. The variant of paragraph 52, wherein the deletion is atpositions corresponding to positions 180 and 181.Paragraph 54. The variant of any of paragraphs 1-53, which furthercomprises a substitution at a position corresponding to position 193.Paragraph 55. The variant of paragraph 54, wherein the substitution at aposition corresponding to position 193 is with Phe.Paragraph 56. The variant of any of paragraphs 1-55, which furthercomprises a deletion at the position corresponding to positions 376and/or 377.Paragraph 57. The variant of any of paragraphs 1-56, which is a variantof a parent alpha-amylase selected from the group consisting of:

a. a polypeptide with at least 60% sequence identity with the maturepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7; or

b. a fragment of the mature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6,or 7, which has alpha-amylase activity.

Paragraph 58. The variant of paragraph 57, wherein the parentalpha-amylase has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, and 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, or7.Paragraph 59. The variant of paragraph 57, wherein the parentalpha-amylase comprises or consists of the amino acid sequence of themature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7.Paragraph 60. The variant of paragraph 57, wherein the parentalpha-amylase is a fragment of the amino acid sequence of the maturepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, wherein the fragmenthas alpha-amylase activity.Paragraph 61. The variant of any of paragraphs 1-60, which is a variantof a parent wild-type alpha-amylase.Paragraph 62. The variant of paragraph 61, wherein the parentalpha-amylase is a Bacillus alpha-amylase.Paragraph 63. The variant of paragraph 62, wherein the parentalpha-amylase is a Bacillus amyloliquefaciens, Bacillus clausii,Bacillus licheniformis, Bacillus stearothermophilus, or Bacillussubtilis alpha-amylase.Paragraph 64. The variant of any of paragraphs 1-63, which has asequence identity of at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100%, to the aminoacid sequence of the parent alpha-amylase.Paragraph 65. The variant of any of paragraphs 1-63, which has asequence identity of at 2 5 least 65%, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, and at least 99%, but less than 100%, with themature polypeptide of SEQ ID NO: 1.Paragraph 66. The variant of any of paragraphs 1-63, which has asequence identity of at 3 0 least 65%, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, and at least 99%, but less than 100%, with themature polypeptide of SEQ ID NO: 2.Paragraph 67. The variant of any of paragraphs 1-63, which has asequence identity of at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, and at least 99%, but less than 100%, with themature polypeptide of SEQ ID NO: 3.Paragraph 68. The variant of any of paragraphs 1-63, which has asequence identity of at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, and at least 99%, but less than 100%, with themature polypeptide of SEQ ID NO: 4.Paragraph 69. The variant of any of paragraphs 1-63, which has asequence identity of at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, and at least 99%, but less than 100%, with themature polypeptide of SEQ ID NO: 5.Paragraph 70. The variant of any of paragraphs 1-63, which has asequence identity of at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, and at least 99%, but less than 100%, with themature polypeptide of SEQ ID NO: 6.Paragraph 71. The variant of any of paragraphs 1-63, which has asequence identity of at least 65%, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, and at least 99%, but less than 100%, with themature polypeptide of SEQ ID NO: 7.Paragraph 72. The variant of any of paragraphs 1-71, wherein the variantconsists of 483 to 515, 483 to 493, or 483 to 486 amino acids.Paragraph 73. A detergent composition comprising a variant of any ofparagraphs 1-72 and a 3 0 surfactant.Paragraph 74. A composition comprising a variant of any of paragraphs1-72 and one or more enzymes selected from the group consisting ofbeta-amylase, cellulase (beta-glucosidase, cellobiohydrolase, andendoglucanase) glucoamylase, hemicellulase (e.g., xylanase), isoamylase,isomerase, lipase, phytase, protease, and pullulanase.Paragraph 75. Use of a variant of any of paragraphs 1-72 for washingand/or dishwashing. Paragraph 76. Use of a variant of any of paragraphs1-72 for desizing a textile.Paragraph 77. Use of a variant of any of paragraphs 1-72 for producing abaked product.Paragraph 78. Use of a variant of any of paragraphs 1-72 for liquefyinga starch-containing material.Paragraph 79. A method of producing liquefied starch, comprisingliquefying a starch-containing material with a variant of any ofparagraphs 1-72.Paragraph 80. A process of producing a fermentation product, comprising

a. liquefying a starch-containing material with a variant of any ofparagraphs 1-72 to produce a liquefied mash;

b. saccharifying the liquefied mash to produce fermentable sugars; and

c. fermenting the fermentable sugars in the presence of a fermentingorganism.

Paragraph 81. The process of paragraph 80, wherein the starch-containingmaterial is liquefied with the variant and a pullulanase, e.g., a GH57pullulanase.Paragraph 82. The process of paragraph 81, wherein the pullulanase isobtained from a strain of Thermococcus, including Thermococcus sp. AM4,Thermococcus sp. HJ21, Thermococcus barophilus, Thermococcusgammatolerans, Thermococcus hydrothermalis; Thermococcus kodakarensis,Thermococcus litoralis, and Thermococcus onnurineus; or from a strain ofPyrococcus, such as Pyrococcus abyssi and Pyrococcus furiosus.Paragraph 83. The process of any of paragraphs 80-82, further comprisingadding a protease, such as an acid fungal protease or a metalloproteasebefore, during and/or after liquefaction.Paragraph 84. The process of paragraph 83, wherein the metalloproteaseis obtained from a strain of Thermoascus, preferably a strain ofThermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No.0670.Paragraph 85. A process of producing a fermentation product, comprisingcontacting a starch substrate with variant of any of paragraphs 1-72, aglucoamylase, and a fermenting organism.Paragraph 86. The process of any of paragraphs 80-85, wherein thefermentation product is selected from the group consisting of alcohol(e.g., ethanol and butanol), organic acids (e.g., succinic acid andlactic acid), sugar alcohols (e.g., glycerol), ascorbic acidintermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).Paragraph 87. An isolated polynucleotide encoding the variant of any ofparagraphs 1-72. Paragraph 88. A nucleic acid construct comprising thepolynucleotide of paragraph 87.Paragraph 89. An expression vector comprising the nucleic acid constructof paragraph 88.Paragraph 90. A host cell comprising the nucleic acid construct ofparagraph 88.Paragraph 91. A method of producing a variant alpha-amylase, comprising:

a. cultivating the host cell of paragraph 90 under conditions suitablefor the expression of the variant; and

b. recovering the variant from the cultivation medium.

Paragraph 92. A transgenic plant, plant part or plant cell transformedwith the polynucleotide of paragraph 87.Paragraph 93. A method for obtaining a variant alpha-amylase, comprising

a. introducing into a parent alpha-amylase a substitution at three ormore (several) positions corresponding to positions 59, 89, 91, 96, 108,112, 129, 157, 165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220,224, 242, 254, 269, 270, 274, 276, 281, 284, 416, and 427, wherein thevariant has at least 65% and less than 100% sequence identity with atleast one of the mature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, and7, and the variant has alpha-amylase activity; and

b. recovering the variant.

What is claimed is:
 1. An isolated variant alpha-amylase, comprising asubstitution at three or more (several) positions corresponding to anyof positions 59, 89, 91, 96, 108, 112, 129, 157, 165, 166, 168, 171,177, 179, 180, 181, 184, 208, 220, 224, 242, 254, 269, 270, 274, 276,281, 284, 416, and 427, wherein the variant has at least 65% and lessthan 100% sequence identity with the mature polypeptide of SEQ ID NO: 1,2, 3, 4, 5, 6, and/or 7 and the variant has alpha-amylase activity. 2.The variant of claim 1, which comprises a substitution at a positioncorresponding to position 59 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, inparticular with Ala, Gln, Glu, Gly, Ile, Leu, Pro, or Thr; asubstitution at a position corresponding to position 89 with Ala, Arg,Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Arg, His, or Lys; a substitution ata position corresponding to position 91 with Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,in particular with Ile, Leu, or Val; a substitution at a positioncorresponding to position 96 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ile, Leu, or Val; a substitution at a positioncorresponding to position 108 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, inparticular with Ala; a substitution at a position corresponding toposition 112 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, in particular with Ala, Asp,or Glu; a substitution at a position corresponding to position 129 withAla, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val, in particular with Ala, Thr, or Val; asubstitution at a position corresponding to position 157 with Ala, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with His, Lys, Phe, or Tyr; asubstitution at a position corresponding to position 165 Ala, Arg, Asn,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val, in particular with Asn; a substitution at a positioncorresponding to position 166 Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val, in particularwith Phe; a substitution at a position corresponding to position 168with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, in particular with Ala; a substitutionat a position corresponding to position 171 with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, orVal, in particular with Glu; a substitution at a position correspondingto position 177 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, in particular with Arg,Leu, or Met; a substitution at a position corresponding to position 179with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, in particular with Gln, Glu, Ile, Leu,Lys, or Val; a substitution at a position corresponding to position 180with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, in particular with Glu or Val; asubstitution at a position corresponding to position 181 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Glu, Gly, or Val; a substitution ata position corresponding to position 184 with Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,in particular with Ser; a substitution at a position corresponding toposition 208 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, in particular with Phe orTyr; a substitution at a position corresponding to position 220 withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val, in particular with Pro; a substitution at aposition corresponding to position 224 with Ala, Arg, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,in particular with Leu; a substitution at a position corresponding toposition 242 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, in particular with Ala, Asp,Glu, Gln, or Met; a substitution at a position corresponding to position254 with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, in particular with Ala, Ser, orThr; a substitution at a position corresponding to position 269 withAla, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val, in particular with Gln or Glu; asubstitution at a position corresponding to position 270 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Leu, Thr, or Val; a substitution ata position corresponding to position 274 with Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,in particular with Arg, Gln, Glu, Lys, or Phe; a substitution at aposition corresponding to position 276 with Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val,in particular with Phe; a substitution at a position corresponding toposition 281 with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, in particular with Asn orSer; a substitution at a position corresponding to position 284 withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro,Ser, Thr, Trp, Tyr, or Val, in particular with His, Thr, or Val; asubstitution at a position corresponding to position 416 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val, in particular with Ala, Asn, Asp, Ser, Thr, or Val;and/or a substitution at a position corresponding to position 427 withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val, in particular with Ile, Met, or Val.
 3. Thevariant of claim 1, comprising a substitution at three positionscorresponding to any of positions 59, 89, 91, 96, 108, 112, 129, 157,165, 166, 168, 171, 177, 179, 180, 181, 184, 208, 220, 224, 242, 254,269, 270, 274, 276, 281, 284, 416, and 427, in particular, at thepositions corresponding to positions 129, 177, and 179 or positions 220,242, and
 254. 4. The variant of claim 1, which has 3-20, e.g., 3-10 and6-10, alterations such as 3, 4, 5, 6, 7, 8, 9 or 10 alterations.
 5. Anisolated variant alpha-amylase, comprising or consisting of a set ofsubstitutions selected from the group consisting of: V59A+G108A;S242Q+M284V; V59A+M284V; G108A+M284V; V59A+G108A+M284V;V59A+G108A+S242Q+M284V; E129V+K177L+R179E; K220P+N224L+Q254S;E129V+K177L+R179E+M284V; V59A+E129V+K177L+R179E+H208Y+M284V;V59A+H208Y+K220P+N224L+Q254S+M284V; E129V+K177L+R179E+K220P+N224L+Q254S;V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S+M284V;V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;V59A+Q89R+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S+M284V; andV59A+G108A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S+M284V;wherein the variant has at least 65% and less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6,and/or 7 and the variant has alpha-amylase activity.
 6. The variant ofclaim 1, which has 3-20, e.g., 3-10 and 6-10, alterations such as 3, 4,5, 6, 7, 8, 9 or 10 alterations.
 7. The variant of claim 6, wherein thealterations are substitutions.
 8. The variant of claim 1, which furthercomprises a deletion at one or more, e.g., two, three or four, positionscorresponding to positions 179, 180, 181 and
 182. 9. The variant ofclaim 1, which further comprises a substitution at a positioncorresponding to position
 193. 10. The variant of claim 1, which furthercomprises a deletion at the position corresponding to positions 376and/or
 377. 11. The variant of claim 1, which is a variant of a parentalpha-amylase selected from the group consisting of: a. a polypeptidewith at least 60% sequence identity with the mature polypeptide of SEQID NO: 1, 2, 3, 4, 5, 6, or 7; or b. a fragment of the maturepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, which hasalpha-amylase activity.
 12. The variant of claim 11, wherein the parentalpha-amylase has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, and 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, or7.
 13. The variant of claim 11, wherein the parent alpha-amylasecomprises or consists of the amino acid sequence of the maturepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, or
 7. 14. The variant ofclaim 11, wherein the parent alpha-amylase is a fragment of the aminoacid sequence of the mature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6,or 7, wherein the fragment has alpha-amylase activity.
 15. The variantof claim 1, which has a sequence identity of at least 65%, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, butless than 100%, to the amino acid sequence of the parent alpha-amylase.16. A detergent composition comprising a variant of claim 1 and asurfactant.
 17. A method of producing liquefied starch, comprisingliquefying a starch-containing material with a variant of claim
 1. 18. Aprocess of producing a fermentation product, comprising a. liquefying astarch-containing material with a variant of claim 1 to produce aliquefied mash; b. saccharifying the liquefied mash to producefermentable sugars; and c. fermenting the fermentable sugars in thepresence of a fermenting organism.
 19. A process of producing afermentation product, comprising contacting a starch substrate withvariant of claim 1, a glucoamylase, and a fermenting organism.